WO2011147138A1 - 靶向性白细胞介素融合蛋白及其制备方法与应用 - Google Patents

靶向性白细胞介素融合蛋白及其制备方法与应用 Download PDF

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WO2011147138A1
WO2011147138A1 PCT/CN2010/076635 CN2010076635W WO2011147138A1 WO 2011147138 A1 WO2011147138 A1 WO 2011147138A1 CN 2010076635 W CN2010076635 W CN 2010076635W WO 2011147138 A1 WO2011147138 A1 WO 2011147138A1
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fusion protein
amino acid
cell
binding region
seq
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French (fr)
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周兵
姜静
周宇
邓磊修
刘武
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山东先声麦得津生物制药有限公司
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to the field of biology, and in particular to an interleukin fusion protein and its preparation and its use in medicine. Background technique
  • Tumor immunotherapy has become one of the most active areas of anti-tumor targeting research, and monoclonal antibodies, adoptive immunotherapy, tumor vaccines, and cytokine therapy have shown complementarity with traditional therapies.
  • Interleukin 2 is an interleukin well known in the art. It was discovered in 1976 that Morgan et al. used plant hemagglutinin to stimulate normal human lymphocytes and found that they can produce a selective A cytokine that promotes the growth of T lymphocytes, which must bind to the IL-2 receptor to exert a biological effect.
  • IL-2 is a variety of biological function cytokines that stimulate cytotoxic T lymphocyte differentiation, activate non-specific killing of cytotoxic T lymphocytes, natural killer cells, eosinophils, mast cells, and enhance B lymphocytes. Antibody secretion and proliferation. Therefore, it is mainly used in the clinical immunization of immune adjuvants to achieve the purpose of adjuvant therapy for tumor control.
  • IL-2 human recombinant IL-2 for the treatment of cancer.
  • IL-2 was approved by the FDA for the treatment of renal cancer and melanoma; IL-2 is also approved for large doses for the treatment of HIV.
  • Clinical trials have shown that IL-2 monotherapy is effective in treating cancer with high side effects.
  • the objective rate of high-dose treatment of melanoma is 5-27%, of which only 0-4% of patients have complete remission.
  • the side effects of IL-2 are large, and the patient has a series of reactions such as body fluid retention, diarrhea, and hypotension. A small number of patients die. These side effects are related to the immune overreaction of other tissues in the whole body caused by IL-2.
  • the strategy mainly includes polyethylene glycol (PEG) modification of IL_2, fusion with human serum albumin (HSA) to extend half-life, glycosylation modification to improve IL-2 stability, and fusion with monoclonal antibodies to target drugs to specific targets. (such as tumor cells), combined with other proteins (such as immunoregulatory factors) to produce a drug combination effect.
  • PEG-modified IL-2 did not significantly improve half-life and efficiency compared to IL-2 and has not been approved by the FDA for cancer treatment.
  • HSA-IL-2 fusion protein Albuleukin Human Genome Sc iences has developed the HSA-IL-2 fusion protein Albuleukin. The plasma half-life of intravenous Albuleukin has increased from 19 minutes to 6 hours in IL-2 and has now entered Phase II clinical trials. The third Thr of natural IL-2 is modified by glycosylation. At present, the glycosylation pathway of IL-2 is divided into eukaryotic cell expression, and synthetic glycosylated short peptide and IL-2 protein linkage strategies. Symans is used to express hcxIL-2 in human 293 cells. Several laboratories in the country have developed yeast-expressing glycosylated IL-2, which is still in the development stage.
  • a possible problem for interleukin-2 coupled/fused with biomacromolecules is that, although the half-life is extended, the multiplication of the molecular weight of the drug makes it more difficult to pass through the physiological barrier to the target tissue, especially for large solid tumors. It makes it difficult to reach the tumor site through the physiological barrier.
  • the lack of specificity and affinity of the targeted drug causes the targeted drug to damage normal cells before reaching the target tissue, consuming drug efficacy.
  • a tumor antigen with high specificity as a target, and also rationally design the targeted drug through protein engineering, reduce the molecular mass, increase the affinity and specificity, increase the concentration of the drug in the tumor tissue, and Reduce the distribution in normal tissues and reduce the toxic side effects on normal cells.
  • Targeted short peptide and non-antibody proteins that are mainly targeted to tumor neovascular endothelial cells and tumor cells, including NGR and integrin (integr ins ov P 3 ) against the surface molecular molecule aminopeptidase of tumor vascular endothelial cells ⁇ RGD.
  • ARENEGYR ARENEGYR
  • Sub-(NGR-hTNF o ) used to treat rectal cancer, liver cancer, mesothelioma has entered Phase I clinical.
  • GE's drug 18F-AH11 1585 (radiolabeled RGD), which was developed for use in tumor diagnosis, also entered Phase I clinical trials.
  • SSTR somatostatin receptor
  • a fusion protein comprising (1) a binding binding region that specifically binds to a tumor cell or a tumor vascular endothelial cell from the N-terminus to the C-terminus; (2) an interleukin.
  • the fusion protein of the present invention may have a junction region of no more than 10 amino acids for ligation of the targeted binding region and interleukin.
  • the junction region has three amino acids: glycine-glycine-serine- (GGS), and in another embodiment, six amino acids: glycine-glycine-serine-glycine-glycine-serine (GGSGGS) .
  • the interleukin in the fusion protein of the present invention is human interleukin 2 having the amino acid sequence of SEQ ID NO: 1.
  • IL-2 Interleukin 2 nucleotide sequence GenBank accession number Li-000586 ⁇ 3.
  • IL-2 is also the first cytokine that has been isolated, purified, and identified at the molecular level. It is a multifunctional secretory monomeric glycoprotein. 5k ⁇ After the cut off the N-terminal 20 hydrophobic amino acid signal peptide, the mature IL-2 is composed of 1 33 amino acids, the molecular weight of 15. 5kD. IL-2 contains three cysteines (Cys 58, Cys 105 and Cys 25), and the first two Cys form an intrachain disulfide bond, which plays an important role in its stability and function.
  • the IL-2 mutant IL-2 C125S is often prepared industrially to promote the correct pairing of its disulfide bonds.
  • the human interleukin 2 in the fusion protein of the invention contains SEQ ID NO: 1 The amino acid sequence in which the cysteine (C) at position U5 is mutated to serine (S).
  • the targeted binding region in the fusion protein of the invention is a human amino acid sequence.
  • the targeted binding region is homologous to somatostatin, and the amino acid sequence of the targeted binding region is not less than 60% homologous to somatostatin.
  • the present invention specifically provides a fusion protein whose amino acid sequence targeting the binding region is FCYWKSCT, as shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO. 2 is substituted, Deletion, or addition of one or several amino acids, is at least 80% identical to the amino acid sequence set forth in SEQ ID NO. 2, and has the activity of specifically binding to tumor cells or tumor vascular endothelial cells.
  • the targeted binding region in the fusion protein of the present invention may comprise no less than
  • the tyrosine in the targeted binding region as shown in SEQ ID NO: 2 is replaced by any amino acid containing a benzene ring or a heterocyclic ring, preferably with phenylalanine or tryptophan, without affecting its target.
  • SEQ ID NO: 2 is replaced by any amino acid containing a benzene ring or a heterocyclic ring, preferably with phenylalanine or tryptophan, without affecting its target.
  • the serine in the targeted binding region as shown in SEQ ID NO: 2 is replaced or deleted by any amino acid-containing amino acid, preferably with threonine, without affecting its targeting.
  • the invention provides a specific fusion protein comprising the amino acid sequence set forth in SEQ ID D NO: 3, designated SIL.
  • an amino acid tag may be attached at the amino (N) terminus or the carboxyl (C) terminus, including but not limited to polyhistidine (poly-His is usually 6 histidine), poly Arginine (Poly-Arg is usually 5-6 arginine) and the like.
  • Another aspect of the invention also provides a DNA molecule encoding a fusion protein of the invention. Due to the homogeneity of the codons, a wide variety of nucleotide sequences can be present which are capable of encoding the specific proteins described herein.
  • the invention provides a DNA molecule encoding a fusion protein comprising an amino acid sequence as set forth in SEQ ID D NO: 3, the nucleotide sequence of which is set forth in SEQ IDD NO:4.
  • the DNA molecule encoding the fusion protein is integrated into an expression cassette, and the expression cassette is then inserted into a suitable expression vector. This expression vector is then transferred to a host cell or animal for recombinant protein expression.
  • the expression cassette includes at least the following contents: (1) a transcription initiation region, (2) can encode the present invention The DNA molecule of the fusion protein, which is carried out under the control of the transcriptional initiation region, and (3) the transcriptional stop region.
  • the transcription initiation region and the transcriptional stop region may be naturally occurring or artificially constructed sequences suitable for transcription of genes in eukaryotic or prokaryotic cells. Such sequences are well known in the art.
  • a suitable DNA fragment containing a transcription initiation sequence and a suitable DNA fragment containing a transcriptional stop region are ligated to a DNA fragment encoding the fusion protein.
  • Such ligation methods are well known in the art, for example, by selecting appropriate restriction endonucleases and ligation.
  • the present invention further provides an expression vector in which a DNA molecule encoding the fusion protein of the present invention is recombined, and a cell expressing the fusion protein and a transgenic animal.
  • the expression vector is preferably a plasmid or a virus.
  • the cell can be a mammalian cell, an insect cell, a yeast or a bacterium.
  • Transgenic animals can be genetically modified sheep, cattle, and so on.
  • an Escherichia coli which recombines a DNA molecule encoding the fusion protein of the present invention, which was deposited on May 24, 2010 in the Chinese Microbial Culture Collection Management Committee. Microbiology Center, deposited under CGMCC No. 3867.
  • the present invention also provides a method for producing the fusion protein, comprising the steps of: providing a cell or a transgenic animal comprising a DNA molecule encoding the fusion protein of the present invention, and expressing the fusion protein to the cell or the transgenic animal; The fusion protein is isolated.
  • the fusion protein of the present invention can enhance the ability of the immune system to kill tumor cells by activating NK cells, CTL cells, and LAK cells in tumor tissues/sites. Therefore, the present invention also provides the use of the fusion protein, that is, The use in the treatment of a disease caused by excessive cell proliferation or in the preparation of a medicament for treating a disease caused by excessive proliferation of cells such as cancer.
  • Figure 1 is a schematic view showing the structure of the fusion protein of the present invention.
  • C human interleukin-2.
  • Fig. 2 shows the results of SDS-PAGE identification of the expression and purification of the fusion protein SIL of the present invention.
  • Lane 1 molecular weight Marker; from top to bottom, 97. 0kDa, 66. 0kDa, 45. OkDa, 30. OkDa, 20. lkDa, 14. 4kDa 0
  • Lane 2 a host lysate expressing the fusion protein
  • Lane 4 Purified target protein by Ni Sepharose High Perf ormance.
  • Figure 3 shows the results of SDS-PAGE of the renaturation of the fusion protein SIL of the present invention.
  • M molecular weight Marker, from top to bottom, 97. OkDa, 66. OkDa, 43. OkDa, 30. OkDa, 20. lkDa, 14. 4kDa;
  • Figure 4 is a diagram showing the reverse phase high performance liquid phase analysis of the fusion protein SIL of the present invention (C18 RP-HPLC), chromatography column: Z0RBA 300SB-C18 5 bacteria, 4. 6*250 mm; detection wavelength: 280 nm; purity: 96.7%.
  • Fig. 5 shows the results of in vitro targeted binding experiments of the fusion protein SIL of the present invention.
  • Figure 6 shows the effect of the fusion protein SIL of the present invention on the proliferation rate of CTLL-2 cells after binding to different target cells.
  • Fig. 7 shows the results of the anti-tumor experiments of the high-dose and low-dose and IL-2 fusion proteins of the fusion protein of the present invention.
  • Figure 8 is a graph showing the comparison of the life extension rates of the fusion proteins S IL and IL-2 of the present invention against Kunming mice inoculated with H22. detailed description
  • the invention discloses an interleukin fusion protein and its preparation and its application in the field of medicine, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is specifically to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the products, methods and applications of the present invention have been described by the preferred embodiments, and it is obvious that those skilled in the art can change or modify and combine the methods and applications described herein without departing from the spirit and scope of the invention.
  • the techniques of the present invention are implemented and applied.
  • a targeted interleukin fusion protein includes (1) a specific binding region for binding to tumor cells or tumor vascular endothelial cells from the N-terminus to the C-terminus; (2) depending on the situation a junction region of no more than 10 amino acids; (3) an interleukin amino acid sequence
  • the targeted binding region in the fusion protein of the present invention may comprise no less than
  • a methionine having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the N-terminus can carry a start codon for E. coli expression, may be heterogeneously cleaved during translation; preferably, will be SEQ.
  • the tyrosine in the targeted binding region shown by ID NO: 2 is replaced by any amino acid containing a benzene ring or a heterocyclic ring, preferably phenylalanine or tryptophan, without affecting its targeting.
  • the tyrosine in the targeted binding region as shown in SEQ ID NO: 2 is replaced or deleted by any of the amino acid-containing amino acids, preferably threonine, without affecting its targeting.
  • the targeted binding regions and interleukins of the fusion proteins of the invention can be joined directly by peptide bonds or by linkage regions.
  • the linking region may have 2 to 10 amino acid linkages to the targeting binding region and interleukin 2, as desired.
  • the junction region has three amino acids: glycine-glycine-serine-(GGS), and in another embodiment, six amino acids, glycine-glycine-serine-glycine-glycine-serine (GGSGGS).
  • the fusion protein of the invention comprises the amino acid sequence set forth in SEQ ID NO: 4, designated S IL.
  • the fusion protein of the invention comprises an amino acid sequence as set forth in SEQ ID NO: 3.
  • Another aspect of the invention also provides a DNA molecule encoding the fusion protein of the invention described above. Due to the homology of the codons, a wide variety of nucleotide sequences ij ij capable of encoding the specific proteins described herein can be present.
  • the invention provides a DNA molecule encoding a fusion protein comprising an amino acid sequence as set forth in SEQ ID D NO: 3, the nucleotide sequence of which is set forth in SEQ IDD NO:4.
  • the DNA molecule encoding the fusion protein is integrated into an expression cassette, and the expression cassette is then inserted into a suitable expression vector. This expression vector is then transferred to a host cell or animal for recombinant protein expression.
  • the expression cassette comprises at least the following contents: (1) a transcription initiation region, (2) a DNA molecule encoding a fusion protein of the present invention, which is carried out under the control of a transcription initiation region, and 3) Transcriptional stop zone.
  • the transcription initiation region and the transcription termination region may be days depending on the host cell used for protein expression. There are, however, artificially constructed sequences that are suitable for transcription of genes in eukaryotic or prokaryotic cells. Such sequences are well known in the art.
  • a suitable DNA fragment containing the transcription initiation sequence is ligated to a suitable DNA fragment containing the transcriptional stop region and a DNA fragment encoding the fusion protein. Such ligation methods are well known in the art, for example, by selecting appropriate restriction endonucleases and ligation.
  • the expression cassette can be inserted into an expression vector which is subsequently transferred into a host cell or animal.
  • the expression vector also includes a replication initiation sequence, as well as a selection marker.
  • the plasmid is a useful vector.
  • plasmid vectors There are a variety of well-known plasmid vectors that can be used for this purpose, including, but not limited to, pET15b, pET22b, pET25b, pET28b, and the like.
  • a yeast expression vector such as pPIC9, pA0815, pPICZ or the like can be used as an expression vector.
  • the expression vector for expression vectors of mammalian cells requires a DNA sequence suitable for integration into a host cell chromosome by homologous recombination.
  • Mammalian cell expression vector such as pcDNA3.1, pSI, and the like.
  • transgenic sheep, cattle, etc. technical means for producing transgenic animals (transgenic sheep, cattle, etc.) containing expression cassettes are well known in the art.
  • pBLG and the like can be used as vectors for expression of transgenic animals.
  • the fusion protein can be expressed under conditions suitable for expression of the fusion protein of the present invention.
  • the fusion protein is isolated and purified by various separation methods by the difference in its physical and chemical properties and other properties. These methods are well known to those skilled in the art, for example: conventional denaturing renaturation treatment, centrifugation, sonication, ultrafiltration membrane filtration, metal affinity chromatography, ion exchange chromatography, gel filtration chromatography, dialysis, high efficiency Conventional purification methods such as liquid chromatography and combinations of these methods.
  • the fusion protein of the present invention enhances the ability of a local tissue and/or an organ immune system to kill tumor cells by activating NK cells, CTL cells, LAK cells, and the like at a tumor tissue/site.
  • Another aspect of the invention also provides a pharmaceutical use of the fusion protein, or a medicament for treating a disease.
  • Example 1 Construction of interleukin 2 fusion protein granules for expression of the somatostatin homologous targeting peptide of the present invention
  • a pair of primers were designed and synthesized based on the N-terminal and N-terminal amino acid sequences of the fusion protein and the N-terminal additional binding region amino acid to be added, and amplified from the human liver cDNA library by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a DNA sequence encoding human interleukin 2 (IL-2) can be obtained, and a DNA sequence encoding human interleukin 2 (C 125 S) can also be directly synthesized as a template, and a pair of primers designed are:
  • upstream primer CATATGTTCTGTTTCTGGAAGACGTGCACCGGCGGTAGCGCACCTAC (as shown in SEQ I D NO: 5)
  • the Ndel site is contained in sequence: CATATG, including a 24-nucleotide targeting binding region coding sequence ⁇ ij : TTCTGTTTCTGGAAG ACGTGCACC , link region sequence: GGCGGT and subsequent interleukin 2 annealing sequence: AGCGCACCTAC;
  • CAACACTGACT CAACACTGACT
  • histidine tag sequence CAACACTGACT
  • the PCR amplification product and the ET25b empty plasmid were digested with Ndel and Hindi II (the tool enzymes were purchased from Takara Co., Ltd., and the plasmid was purchased from Novage).
  • the above-mentioned fragments were all recovered from agarose gel to recover fragments of corresponding size.
  • the T4 DNA ligase was ligated to each fragment, and the resulting recombinant plasmid was an expression vector for the interleukin fusion protein, and was named pET-S-IL.
  • the structure of the fusion protein is shown in Fig. 1.
  • the ligation reaction mixture was transformed into E. coli XL-lBlue, and after incubation in a medium containing penicillin overnight, positive colonies were selected and DNA was prepared therefrom, and the nucleotide sequence was as shown in SEQ ID NO: 4 by DNA sequence analysis. .
  • the recombinant plasmid was transformed into a competent E. coli BL21 (DE3) strain (Novagen) carrying the T7 promoter gene, which is an engineered strain expressing the corresponding protein, and named as S IL-BL2 L.
  • E. coli BL21 DE3 strain (Novagen) carrying the T7 promoter gene, which is an engineered strain expressing the corresponding protein, and named as S IL-BL2 L.
  • Example 2 Expression and purification of the fusion protein of the present invention
  • the E. coli BL21 clone transformed with the recombinant plasmid pET-S-IL was added to a culture flask of LB medium (37 °C), and the bacterial density reached OD600 « 4-6. 5L fermentation was carried out according to the inoculum amount of 5-10%. Pot culture, to OD600 « 10-20, add 0. 5-lmM isopropyl- ⁇ _D-thiogalactoside to the medium (IPTG) was induced to express, and after induction, OD600 no longer increased the lower can, and the cells were harvested by centrifugation at 10,000 gx (15 minutes).
  • the harvested bacteria were suspended in 3 M urea, 100 mM phosphoric acid, 20 mM Tr i s pH 8. 0 buffer.
  • the eluate containing the fusion protein of interest eluted from the hydrazine affinity column as described above was rapidly diluted in a volume ratio of 1:10 to contain 3 M urea, 100 mM NaCK 20 mM Tris-HCl (pH 8. 0 , 5 mM reduced glutathione and 0.05 mM oxidized glutathione solution to refold (refold) the protein. After 48 hours at 4 degrees, it was dialyzed into PBS and concentrated with an ultrafiltration membrane having a molecular weight cut off of 5,000 daltons for protein purity analysis, as shown in Figs. 3 and 4.
  • Example 3 In vitro targeting of the fusion protein of the present invention
  • SIL interleukin 2 and the fusion
  • CTLL-2 cells in the negative control wells were basically apoptotic, 5 mg/ml was added per well.
  • the tetramethylthiazole blue (MTT) was about 10 ul, and the effect was about 4 h.
  • SDS-HC1 lysate was added overnight, and the 0D value was measured at 570 nm by a microplate reader to calculate the CTLL-2 proliferation rate.
  • Kunming mice were inoculated subcutaneously in the right forelimb of mice with liver cancer (H22), inoculated with lxl 0 6 /ml, 0.2 ml / only, divided into normal saline group, IL-2 high dose group (6000U / only / day), Low IL (3000 U/day/day), high SIL (6000 U/day/day), low SIL (3000 U/day/day), 10 in each group, intravenous administration for 12 days after tumor implantation . After stopping the drug for 6 days, the mice were sacrificed, the tumors were stripped and weighed, and the average tumor type and tumor inhibition rate of each group were calculated. The tumor inhibition rate is calculated by the following formula. Average tumor weight of the control group - mean tumor weight of the drug-administered group
  • Kunming mice were intraperitoneally inoculated with mouse liver cancer (H22), inoculated with 5 x l 0 6 /ml, 0.2 ml / only, divided into normal saline group, IL-2 high dose group (10000 U / only / day), IL (5000 U/day/day), low IL (2500 U/day/day), SIL high (10000 U/day/day), SIL (5000 U/day/day,) SIL low (2500 U/only/ Days, 10 in each group, intravenous administration for 14 days after tumor implantation. Continue to observe after stopping the drug, record the survival and death of each group of mice.
  • the IL-2 fusion protein linked to the targeted binding region homologous to somatostatin of the present invention can specifically bind to various tumor cells such as human gastric cancer, liver cancer, breast cancer and pancreatic cancer, and Dose-related, significantly enhances the targeting of IL-2. After enrichment into tumor tissue, it reduces the toxic side effects on other tissues of the body and SIL is enriched in the tumor site, which greatly enhances the induction of CTL cells to kill tumor cells. effect.
  • SIL is enriched in the tumor site, which greatly enhances the induction of CTL cells to kill tumor cells. effect.

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Description

靶向性白细胞介素融合蛋白及其制备方法与应用
本申请要求于 2010 年 5 月 28 日提交中国专利局、 申请号为 201010185503.2、 发明名称为"靶向性白细胞介素融合蛋白及其制备方法与应 用 "的中国专利申请的优先权， 其全部内容通过引用结合在本申请中。 技术领域
本发明涉及生物领域， 具体涉及白细胞介素融合蛋白及其制备和其在医 药领 i或的应用。 背景技术
传统治疗癌症的方法有手术治疗、 化学治疗、 放射治疗和激素治疗等。 近 年来， 开发选择性作用于特定靶点的高效、 低毒、 特异性强的抗肿瘤分子靶向 药物已成为重要研究开发方向。肿瘤免疫治疗已成为最活跃的抗肿瘤靶向研究 领域之一， 单克隆抗体、 细胞过继免疫治疗、 肿瘤疫苗、 细胞因子治疗已显示 出与传统疗法的互补性。
白细胞介素 2 ( IL-2 )是本领域熟知的一种白细胞介素， 发现于 1976年， Morgan 等人用植物的血球凝集素刺激正常的人淋巴细胞， 发现其可以产生一 种能选择性促进 T淋巴细胞生长的细胞因子， 它必须和 IL-2受体结合才能发 挥生物学效应。 IL-2是多种生物功能细胞因子， 可刺激细胞毒 T淋巴细胞分 化、 激活细胞毒 T淋巴细胞、 自然杀伤细胞、 嗜酸性细胞、 肥大细胞的非特异 性杀伤功能， 也能增强 B淋巴细胞的抗体分泌和增殖。 因此临床上也主要用于 免疫佐剂调动机体的系统免疫， 达到辅助治疗控制肿瘤的目的。
作为第一个被分离纯化的白细胞介素分子， 80年代美国 Cetus公司开发 人源重组 IL-2用于治疗癌症， 直至 1993年， IL-2被 FDA批准用于治疗肾癌 和黑色素瘤； 目前 IL-2也被批准大剂量用于治疗 HIV。 临床试验表明 IL-2单 药治疗癌症的有效率低， 副作用大。 高剂量治疗黑色素瘤的客观有效率为 5-27%, 其中只有 0-4%的病人病情完全緩解。 IL-2的副作用大， 病人产生体液 储留、 腹泻、 血压过低等一系列反应， 小部分患者死亡， 这些毒副作用多与 IL-2引发非治疗病灶的全身其他组织免疫过度反应有关。
针对于 IL-2临床使用的局限， 目前 IL-2蛋白质药物研究主要方向是减小 药物副作用、提高药效、延长药物半衰期。策略主要包括 IL_2 的聚乙二醇（PEG ) 修饰、与人血清白蛋白（HSA )融合延长半衰期，糖基化修饰提高 IL-2稳定性， 与单克隆抗体融合将药物靶向到特定靶点（如肿瘤细胞）， 与其他蛋白（如免疫 调节因子）融合产生药物联用效果等。在临床实验中， PEG修饰的 IL-2与 IL-2 相比， 并没有显著改善半衰期和有效率， 至今未被 FDA批准用于癌症治疗。 Human Genome Sc iences公司开发了 HSA-IL-2融合蛋白 Albuleukin, 静脉注 射 Albuleukin 的血浆半衰期从 IL-2的 19分钟提高到 6小时， 目前已进入二 期临床实验。 天然 IL-2的第三位 Thr是被糖基修饰的， 目前 IL-2的糖基化地 手段分为真核细胞表达、 以及合成的糖基化短肽和 IL-2蛋白连接两种策略， Symans i s公司用人 293细胞表达 hcxIL-2 已经在市面上出售， 国内有若干个 实验室都开发酵母表达糖基化的 IL-2 , 目前还处于开发阶段。 目前默克公司 将抗神经节苷脂 GD2的 C端融合 IL-2的抗体药物 EMD273066 (hul4. 18-IL-2) 用于治疗黑色素瘤已经完成 I期临床， 结果表明 33例病人中， 8例病情稳定。 无 4级毒性反应（死亡）发生， 但仍发现有低血压、 组织缺氧等 3级毒性反应。 此外也有将 IL-2融于其他抗体（如 ant i_her2 ant ibody, ant i-her 3 , IgG3等) 的研究， 以增强 IL-2诱导的 T细胞对肿瘤细胞杀伤特异性。 其他 IL-2融合蛋 白药物， 如 Aerolys in_IL_2 , IL-6-IL-2 , GCSF-IL-2 , 重组人 α胸腺素原- 白介素 2等， 产生药物联用的效果。
对于与生物大分子耦联 /融合的白介素 -2可能存在的问题是， 虽然半衰期 延长但药物分子量的成倍增加使其更难于穿过生理屏障到达靶组织，尤其对大 型实体瘤的浸润性差， 使其难于穿过生理屏障到达肿瘤部位。 靶向药物的特异 性和亲和性不高会造成靶向药物在到达靶组织以前损伤正常细胞， 消耗了药 效。
针对上述问题， 首先要选择特异性高的肿瘤抗原为靶点， 另外还要通过蛋 白质工程对靶向药物进行合理设计， 减小分子质量， 提高亲和力和特异性， 增 加药物在肿瘤组织的浓度和减少在正常组织的分布，减少对正常细胞的毒副作 用。可以选择主要靶向于肿瘤新生血管内皮细胞和肿瘤细胞的靶向短肽及非抗 体蛋白，其中有针对肿瘤新生血管内皮细胞表面分子氨基肽酶 Ν的 NGR和整联 蛋白（integr ins o v P 3 々 RGD。 某些利用上述的相关靶向肽的开发药物， 已 经成功进入临床实验。如 Modmed公司开发 ARENEGYR就为 NGR融合肿瘤坏死因 子（NGR-hTNF o ) , 用于治疗直肠癌、 肝癌、 间皮瘤已经进入 1 1期临床。 GE公 司开发用于肿瘤诊断的药物 18F-AH11 1585 (放射性标记的 RGD)也进入一期临 床。
生长抑素（ Soma tos ta t in, SST )及生长抑素类似物如伐普肽（Vapreot ide) 和兰瑞肽（Lanreot i de)等作为单药已经分别成功用于心血管疾病（脑血栓等） 和类癌、 胰高血糖素瘤、 胃泌素瘤等的临床治疗。 生长抑素受体（SSTR )发现 表达于多种肿瘤细胞， 这些研究充分支持了靶向肽选择的合理性和可行性。 发明内容
本发明的目的是提供一种靶向性白介素融合蛋白，该融合蛋白能够特异性 结合某些细胞特别是肿瘤组织细胞或其新生血管内皮细胞，显著的增加白细胞 介素在靶向组织的浓度， 降低其在其他组织的浓度， 从而提高治疗效果， 降低 毒副作用。
为了实现上述发明目的， 本发明采用以下技术方案：
一种融合蛋白， 从 N端至 C端包括（ 1 )特异性结合肿瘤细胞或者肿瘤血 管内皮细胞的靶向结合区； （2 ) 白细胞介素。
根据情况， 本发明所述融合蛋白可有不多于 10个氨基酸组成的连接区， 用于连接所述靶向结合区和白细胞介素。
在本发明的一个实施方案中， 连接区有三个氨基酸： 甘氨酸-甘氨酸 -丝氨 酸- ( GGS ), 在另一个实施例中有 6个氨基酸： 甘氨酸 -甘氨酸-丝氨酸-甘氨酸 -甘氨酸-丝氨酸（ GGSGGS )。
作为优选， 本发明所述融合蛋白中白细胞介素为人源白细胞介素 2 , 具有 如 SEQ ID NO: 1的氨基酸序列。
白细胞介素 2 ( IL-2 )核苷酸序列 GenBank登录号丽—000586· 3。 IL-2 也是最早在分子水平被分离、 纯化、 鉴定的细胞因子。 它是具有多功能的分泌 单体糖蛋白。切去 N端 20个疏水氨基酸的信号肽后， 成熟 IL-2为 1 33个氨基 酸组成，分子量为 15. 5kD。 IL-2含有三个半胱氨酸（Cys 58、 Cys l 05和 Cys l 25) , 前两个 Cys形成链内二硫键， 对其稳定性和功能发挥具有重要作用。 因此工业 上常制备 IL-2突变体 IL-2 C125S , 以促进其二硫键的正确配对。 在一种实施 方案中， 本发明所述融合蛋白中的人源白细胞介素 2含有如 SEQ I DD N0: 1所 述的氨基酸序列，其中第 U5位半胱氨酸（C ) 突变为丝氨酸（S )。
作为优选， 本发明所述融合蛋白中靶向结合区为人源氨基酸序列。
更优选地， 所述靶向结合区与生长抑素同源， 所述靶向结合区的氨基酸 序列与生长抑素同源性不低于 60%。
在具体实施方式中， 本发明具体提供一种融合蛋白， 其靶向结合区的氨基 酸序列为 FCYWKSCT, 如 SEQ ID NO: 2所示， 或为 SEQ ID NO. 2所示的氨基酸 序列经过取代、 缺失、 或添加一个或几个氨基酸衍生的与 SEQ ID NO. 2所示 的氨基酸序列至少有 80%的一致性， 且具有特异结合肿瘤细胞或者肿瘤血管内 皮细胞的活性。
在一种实施方案中，本发明所述融合蛋白中靶向结合区可以是包括不少于
3个但不多于 50个氨基酸的氨基酸序列， 优选的序列含有生长抑素同源的不 多于 15个氨基酸序列， 在一个优选的实施方案中， 本发明所述融合蛋白的靶 向结合区含有如 SEQ ID NO: 2所述的氨基酸序列， 其 N末端可带有大肠杆菌 表达的起始密码子翻译的甲硫氨酸， 在翻译过程中可能被不均一切除。
作为优选， 将如 SEQ ID NO: 2所示的靶向结合区中酪氨酸由任一含苯环或 杂环的氨基酸替换， 优选替换为苯丙氨酸或色氨酸， 不影响其靶向性。
作为优选， 将如 SEQ ID NO: 2所示的靶向结合区中丝氨酸由任一含有羟基 的氨基酸替换或缺失， 优选替换为苏氨酸， 不影响其靶向性。
在一个优选实施方案中，本发明提供一种具体的融合蛋白，含有如 SEQ IDD N0: 3所述的氨基酸序列，记作 SIL。为了使融合蛋白质便于纯化，可在氨基（ N ) 末端或羧基 ( C )末端连接上氨基酸标签， 包括但不限于多聚组氨酸（ Poly-Hi s 通常为 6个组氨酸）、 多聚精氨酸（Poly-Arg通常为 5-6个精氨酸）等。
本发明的另一方面也提供了编码本发明所述融合蛋白的 DNA分子。由于密 码子的筒并性， 可以存在很多种能够编码本发明所述的特定蛋白的核苷酸序 歹l。 在一个实施方案中， 本发明提供了可以编码含有如 SEQ IDD N0: 3所述氨 基酸序列的融合蛋白的 DNA分子， 其核苷酸序列如 SEQ IDD N0: 4所示。
为了生产本发明的融合蛋白， 编码融合蛋白的 DNA 分子被整合至表达盒 中， 随后表达盒被插入合适的表达载体。 然后， 这一表达载体被转入宿主细胞 或者动物用于重组蛋白表达。
表达盒至少包括以下几个内容： （ 1 )转录起始区， （ 2 )可以编码本发明所 述的融合蛋白的 DNA分子，该转录是在在转录起始区的调控下进行的，以及（ 3 ) 转录中止区。
根据用于蛋白表达的宿主细胞的不同，转录起始区和转录中止区可以是天 然存在或者人工构建的序列， 该序列适合于真核或者原核细胞中的基因转录。 此类序列在本技术领域内已为人所熟知。适当的含有转录起始序列的 DNA片与 适当的含有转录中止区的 DNA片段与编码融合蛋白的 DNA片段相连接。这种连 接方法在本技术领域内已为人所熟知， 比如，选择适当的限制性内切酶酶切以 及连接。
本发明进一步提供重组了编码本发明所述融合蛋白的 DNA分子的表达载 体以及表达所述融合蛋白的细胞和转基因动物。 所述表达载体优选质粒或病 毒。 所述细胞可以是哺乳动物细胞， 昆虫细胞， 酵母或者细菌。 转基因动物可 以是转基因羊， 牛， 等等。
在本发明的实施例中， 具体提供一种重组了编码本发明所述的融合蛋白 的 DNA分子的大肠杆菌，所述大肠杆菌于 2010年 5月 24 日保藏在中国微生物 菌种保藏管理委员会普通微生物中心， 保藏编号为 CGMCC No. 3867。
另外， 本发明也提供了所述融合蛋白的生产方法， 包括以下步骤： 提供 含有编码本发明所述的融合蛋白的 DNA分子的细胞或转基因动物，使该细胞或 转基因动物表达所述融合蛋白以及分离所述融合蛋白。
实验表明，本发明所述融合蛋白可通过在肿瘤组织 /部位激活 NK细胞、 CTL 细胞、 LAK细胞， 提高免疫系统杀伤肿瘤细胞的能力， 因此本发明还提供了所 述融合蛋白的应用，即用于治疗细胞过度增生导致的疾病或制备治疗细胞过度 增生导致的疾病如癌症的药物中的用途。 生物保藏说明
分类命名： 大肠埃希氏菌， ^ e '^ 'a co '.于 2010年 5月 24 日保藏 在中国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区北辰 西路 1号院 3号， 中国科学院微生物研究所， 保藏编号为 CGMCC No. 3867。 附图说明：
图 1示本发明所述融合蛋白结构示意图。 A: 靶向结合区； B: 连接区； C: 人源白细胞介素 2。
图 2示本发明所述融合蛋白 SIL表达与纯化后的 SDS-PAGE鉴定结果。
泳道 1 :分子量 Marker;从上至下依次为 97. 0kDa , 66. 0kDa、 45. OkDa , 30. OkDa , 20. lkDa , 14. 4kDa0
泳道 2: 表达融合蛋白的宿主菌裂解物；
泳道 3: 最终洗涤的包涵体；
泳道 4: 经 Ni Sepharose High Perf ormance亲合纯化的目的蛋白。
图 3 示本发明所述融合蛋白 SIL复性的 SDS-PAGE结果。
A: 15% SDS-PAGE 分析， 0:非还原电泳；
M:分子量 Marker , 从上至下依次为 97. OkDa、 66. OkDa , 43. OkDa , 30. OkDa , 20. lkDa , 14. 4kDa;
R: SIL还原电泳；
图 4示本发明所述融合蛋白 SIL碳 18反相高效液相分析图 （C18 RP-HPLC ) ， 层析柱： Z0RBA 300SB-C18 5菌， 4. 6*250mm; 检测波长： 280nm; 纯度： 96. 7%。 图 5本发明所述融合蛋白 SIL体外靶向结合实验结果。
图 6示本发明所述融合蛋白 SIL结合不同靶细胞后对 CTLL-2细胞增殖率影响。 图 7示本发明所述融合蛋白 SIL高剂量与低剂量与 IL-2体内抗肿瘤实验对比 结果。
图 8示本发明所述融合蛋白 S IL与 IL-2对接种 H22昆明小鼠生命延长率对比。 具体实施方式
本发明公开了一种白细胞介素融合蛋白及其制备和其在医药领域的应 用， 本领域技术人员可以借鉴本文内容， 适当改进工艺参数实现。 特别需要指 出的是， 所有类似的替换和改动对本领域技术人员来说是显而易见的， 它们都 被视为包括在本发明。本发明的产品、 方法及应用已经通过较佳实施例进行了 描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法 和应用进行改动或适当变更与组合， 来实现和应用本发明技术。
依照本发明 ,一种靶向性的白细胞介素的融合蛋白，从 N端至 C端包括（ 1 ) 特异性结合肿瘤细胞或者肿瘤血管内皮细胞的靶向结合区；（2 )根据情况可有 一不多于 10个氨基酸的连接区； （ 3 ) 白细胞介素氨基酸序列 在一种实施方案中，本发明所述融合蛋白中靶向结合区可以是包括不少于
3个但不多于 50个氨基酸的氨基酸序列， 优选的序列含有生长抑素同源的不 多于 15个氨基酸序列， 在一个优选的实施方案中， 本发明所述融合蛋白的靶 向结合区含有如 SEQ ID NO: 2所述的氨基酸序列， 其中 N端可带有大肠杆菌 表达的起始密码子翻译的甲硫氨酸，在翻译过程中可能被不均一切除； 作为优 选， 将如 SEQ ID NO: 2所示靶向结合区中酪氨酸由任一含有苯环或杂环的氨基 酸替换， 优选苯丙氨酸或色氨酸， 不影响其靶向性。
作为优选， 将如 SEQ ID NO: 2所示靶向结合区中酪氨酸由任一含有羟基的 氨基酸替换或缺失， 优选为苏氨酸， 不影响其靶向性。
本发明所述融合蛋白的靶向结合区和白细胞介素可以直接由肽键或由连 接区连接到一起。 视需要而定， 连接区可以有 2至 1 0个氨基酸连接靶向结合 区与白细胞介素 2。
在一个实施方案中，连接区有三个氨基酸：甘氨酸-甘氨酸-丝氨酸 _( GGS ), 在另一个实施例中有 6 个氨基酸甘氨酸-甘氨酸 -丝氨酸-甘氨酸-甘氨酸 -丝 氨酸（ GGSGGS )。
在一个优选实施方案中， 本发明所述融合蛋白含有如 SEQ IDD NO: 4所述 的氨基酸序列， 记作 S IL。
在一个优选实施方案中， 本发明所述融合蛋白含有如 SEQ IDD NO: 3所述 的氛基酸序列。
本发明的另一方面也提供了编码本发明上述的融合蛋白的 DNA分子。由于 密码子的筒并性，可以存在很多种能够编码本发明所述的特定蛋白的核苷酸序 歹 ij。 在一个实施方案中， 本发明提供了可以编码含有如 SEQ IDD N0: 3所述氨 基酸序列的融合蛋白的 DNA分子， 其核苷酸序列如 SEQ IDD N0: 4所示。
为了生产本发明的融合蛋白， 编码融合蛋白的 DNA 分子被整合至表达盒 中， 随后表达盒被插入合适的表达载体。 然后， 这一表达载体被转入宿主细胞 或者动物用于重组蛋白表达。
表达盒至少包括以下几个内容： （ 1 )转录起始区， （ 2 )可以编码本发明所 述的融合蛋白的 DNA分子，该转录是在在转录起始区的调控下进行的，以及（ 3 ) 转录中止区。
根据用于蛋白表达的宿主细胞的不同，转录起始区和转录中止区可以是天 然存在或者人工构建的序列， 该序列适合于真核或者原核细胞中的基因转录。 此类序列在本技术领域内已为人所熟知。适当的含有转录起始序列的 DNA片与 适当的含有转录中止区的 DNA片段与编码融合蛋白的 DNA片段相连接。这种连 接方法在本技术领域内已为人所熟知， 比如， 选择适当的限制性内切酶酶切以 及连接。
表达盒可以被整合插入表达载体，随后这一表达载体被转入宿主细胞或者 动物。 一般情况下， 表达载体还要包括复制起始序列， 以及筛选标记， 例如， 对于细菌的蛋白表达，质粒是一种很有用途的载体。本技术领域内有很多种为 人所熟知的可以用于此目的的质粒载体，包括，但是不仅限于，pET15b、pET22b、 pET25b、 pET28b 等等。 如果通过酵母细胞来重组表达融合蛋白， 酵母表达载 体， 比如 pPIC9 , pA0815 , pPICZ等等， 可以作为表达载体。 如果通过哺乳动 物细胞来进行蛋白表达，也有很多合适的重组蛋白表达载体。用于哺乳动物细 胞表达蛋白的表达载体包含的 DNA序列，需要适合以同源重组方式整合插入宿 主细胞染色体。 哺乳动物细胞表达载体， 比如 pcDNA3. l，pSI等等。 对于通过 动物来表达融合蛋白， 用于生产含有表达盒的转基因动物（转基因羊， 牛， 等 等）的技术手段， 在本技术领域内已为人所熟知。 比如 pBLG等等，可以作为转 基因动物表达的载体。
获得转化的宿主细胞或者转基因动物后，可在适合表达本发明融合蛋白的 条件下表达出融合蛋白。通过其理化性质和其它特性的差别运用各种分离方法 分离纯化融合蛋白。 这些方法是本领域技术人员所熟知的， 例如： 常规的变性 复性处理、 离心、 超声破碎、 超滤膜过滤、 金属亲和层析、 离子交换层析、 凝 胶过滤层析、 透析、 高效液相层析等常规纯化方法及这些方法的结合。
本发明所述融合蛋白通过在肿瘤组织 /部位激活 NK细胞、 CTL细胞、 LAK 细胞等提高局部组织和 /或器官免疫系统杀伤肿瘤细胞的能力。 本发明的另一 个方面还提供所述融合蛋白的医药用途， 或可以生产用于治疗一种疾病的药 物。
下面结合实施例， 进一步阐述本发明。 实施例 1. 用于表达本发明所述生长抑素同源靶向肽的白介素 2融合蛋 白质粒的构建 基于融合蛋白的 N末端和 N末端氨基酸序列以及待加入之 N末端附加的 靶向结合区氨基酸， 设计并合成一对引物， 并以聚合酶链反应 （PCR )技术从 人肝脏 cDNA文库中扩增得到编码人白细胞介素 2 ( IL-2 )的 DNA序列，也可以 直接合成编码人白细胞介素 2 (C 125 S)的 DNA序列作为模板， 设计的一对引物 为：
5上游引物： CATATGTTCTGTTTCTGGAAGACGTGCACCGGCGGTAGCGCACCTAC (如 SEQ I D NO: 5所示 )
其中依次含有 Ndel位点： CATATG , 包括 24个核苷酸的靶向结合区编码序 歹 ij : TTCTGTTTCTGGAAG ACGTGCACC , 链接区序列： GGCGGT以及其后的白细胞介 素 2退火序列： AGCGCACCTAC;
3 (下游）引物： CAACACTGACTCACCATCACCATCACCATTGAAGCTT (如 SEQ ID NO: 6 所示）
其中依次含有退火序列： CAACACTGACT、 组氨酸标签序列：
CACCATCACCATCACCAT, 终止密码子 TGA及其后的 H ind l l l位点。
以 Ndel和 H indi I I酶切 PCR扩增产物以及 ET25b空质粒（（工具酶皆购 自 Takara公司， 质粒购自 Novage公司 ), 上述酶切片段均以琼脂糖凝胶回收 相应大小的片段， 用 T4 DNA连接酶连接各片段， 所得的重组质粒即为白细胞 介素融合蛋白的表达载体， 命名为 pET-S-IL。 融合蛋白结构如图 1所示。
首先将连接反应混合物转化大肠杆菌 XL-lBlue , 在含有青霉素的培养基 中保温过夜后， 挑选阳性菌落并从中大量制备 DNA, 经 DNA序列分析， 其核苷 酸序列如 SEQ I D NO: 4所示。
进一步证实序列正确性后， 将此重组质粒转化到携带 T7启动子基因的感 受态大肠杆菌 BL21 ( DE3 ) 菌株（Novagen ) 中， 即为表达相应蛋白的工程菌， 命名为 S IL-BL2 L 实施例 2: 本发明所述融合蛋白的表达与纯化
将重组质粒 pET-S-IL转化的大肠杆菌 BL21克隆加在 LB培养基的培养瓶 中培养（ 37 °C ) , 待细菌密度达到 OD600 « 4-6 , 按接种量 5-10%进行 5L发酵罐 培养，待 OD600 « 10-20,向培养基中加入 0. 5-lmM异丙基- β _D -硫代半乳糖苷 ( IPTG )进行诱导表达，至诱导后 OD600 不再增加下罐，以 10， 000 g x ( 15分 钟） 离心收获菌体。
将收获的菌体重悬于 3M尿素 l OOmM磷酸 20mM Tr i s pH8. 0緩沖液
( buffer ) 中， 室温放置 1小时， 并将细胞超声处理 3次， 20, 000 g离心收 集包涵体。 然后将包涵体溶解于 8M尿素 l OOmM磷酸钠 50mM巯基乙醇 Tr i s ( H 8. 0 )緩沖中放置 4小时， 以 20000g离心 20分钟收集上清。
将上清液上样于用上述緩沖液预平衡过的 Ni Sepharose High Performance ( GE公司）亲合柱， 用 8M尿素 l OOmM磷酸钠 20mM咪唑緩沖液 以 pH 8. 0-4. 0进行 pH递减洗脱， pH4. 0时得到纯度大于 90%的重组蛋白，如图 2所示， 其氨基酸序列如 SEQ ID NO: 3所示。
将按上述方法从 ΝΓ亲合柱洗脱下来的含目的融合蛋白的洗脱物， 按 1: 10 的体积比迅速稀释于含有 3M尿素、 l OOmM NaCK 20mM Tr i s-HCl ( pH 8. 0 )、 5mM还原态谷胱甘肽和 0. 05mM氧化态谷胱甘肽的溶液中以使蛋白质重折叠 (复性）。 经 4度放置 48小时后透析于 PBS中， 并用截留分子量为 5000道尔 顿的超滤膜浓缩， 进行蛋白纯度分析， 如图 3和图 4所示。 实施例 3: 本发明所述融合蛋白体外靶向性研究
将人胃癌细胞 SGC-7901 , 人肝癌细胞 SMMC-7721 , 人乳腺癌细胞 MB-MDA-23K人胰腺癌细胞 PANC-1、人脐静脉内皮细胞 HUVEC、 人胚胎成纤维 细胞 M-20体外常规培养， 消化离心， 将上述细胞分别接种到 12孔细胞培养板 中，待细胞长至培养板 70%-80%左右，加入不同浓度的白细胞介素 2及本发明 所述融合蛋白 S-白介素 2 (SIL)样品 （终浓度分别为 2000 U/mK 1000 U/ml 、 500U/ml ), 设 PBS阴性对照组， 加药后作用约 4h后弃上清， 用 PBS连续清洗 三遍， 沖洗干净后加入无 IL_2、 密度 20 X lOVml的小鼠 T淋巴细胞（ CTLL-2 ) 细胞 lml , 过夜培养， 第二天显微镜下观察， 待阴性对照孔 CTLL-2细胞基本 凋亡时， 每孔加入 5mg/ml的四甲基噻唑蓝（MTT )约 l OOul , 作用约 4h, 加入 SDS-HC1裂解液过夜， 酶标仪 570nm下测 0D值， 计算 CTLL-2增殖率。
计算公式： CTLL-2增殖率 = ( 0D 加药 ft/0D ^组- 1 ) X 100%
结果表明： 无靶向结合区的白细胞介素 2 ( IL-2 ) 与人胃癌细胞 ( SGC-7901 ) 不结合， SIL能与之结合且呈剂量相关性， MTT显色结果与显微 镜下观察结果一致（如图 5 ); SIL与人胃癌、 肝癌、 乳腺癌、 胰腺癌等肿瘤细 胞有特异性结合， 与内皮细胞略有结合， 且呈剂量相关性， 与胎儿成纤维细胞 基本不能结合（如图 6 ), 说明多种肿瘤细胞及内皮细胞表达生长抑素受体， 虽有差异但融合蛋白的靶向结合区可与之结合。 实施例 4: SIL体内抗肿瘤活性——抑瘤率实验
昆明小鼠右前肢腋下接种小鼠肝癌 （H22 ), 按 l x l 06/ml , 0. 2ml/只进行 接种， 分为生理盐水组、 IL-2高剂量组（ 6000U/只 /天）、 IL低（ 3000 U/只 / 天）、 SIL高 （ 6000 U/只 /天）、 S IL低（ 3000 U/只 /天）， 每组 10只， 接瘤后 静脉给药连续给药 12天。 停药后观察 6天， 处死小鼠， 剥离肿瘤称重， 计算 每组平均瘤种和抑瘤率。 抑瘤率以下式计算。 对照组平均瘤重-给药组平均瘤重
抑瘤率 （％) = X 100%
对照组平均瘤重 结果表明，在高剂量组， SIL与 IL-2的抑瘤率对比，无显著差异（ P>0. 05 ), 在低剂量组， SIL与 IL的抑瘤率对比， 有非常显著差异（P<0. 01 ), 结果如图 7所示； IL-2高剂量组有两只动物死亡， SIL高剂量动物没有死亡， 说明 SIL 富集到肿瘤组织后，降低了对全身其它组织的毒副作用且 SIL富集到肿瘤部位 后， 大大增强了其诱导 CTL细胞杀伤肿瘤细胞的作用。 实施例 5: SIL体内抗肿瘤活性 __生命延长率实验
昆明小鼠腹腔接种小鼠肝癌 （H22 ), 按 5 x l 06/ml , 0. 2ml/只进行接种, 分为生理盐水组、 IL-2高剂量组（ 10000U/只 /天）、 IL中 （ 5000 U/只 /天）、 IL低（ 2500 U/只 /天）、 SIL高 （ 10000 U/只 /天）、 SIL中 （ 5000 U/只 /天、） SIL低（ 2500 U/只 /天）， 每组 10只， 接瘤后静脉给药连续给药 14天。 停药 后继续观察， 记录各组小鼠生存及死亡情况。
结果表明， 截至实验结束（40天）， 生理盐水组动物均已死亡， 其它各给 药组存活动物只数分别为： SIL高（ 9只）〉 SIL中（ 6只）〉 IL高（ 5只） =SIL 低（5只）〉 IL中 （4只） = IL低（4只 ) , S IL各剂量组存活小鼠数目均多于 IL各剂量组， 说明增加 IL-2的靶向性后， 明显增强了其抗肿瘤的活性， 结果 如图 8所示。 以上结果说明， 本发明所述连接了与生长抑素同源的靶向结合区的 IL-2 融合蛋白可与人胃癌、 肝癌、 乳腺癌、 胰腺癌等多种肿瘤细胞特异性结合， 且 呈剂量相关性， 显著增强 IL-2的靶向性， 富集到肿瘤组织后， 降低了对全身 其它组织的毒副作用且 SIL富集到肿瘤部位后，大大增强了其诱导 CTL细胞杀 伤肿瘤细胞的作用。 以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通 技术人员来说， 在不脱离本发明原理的前提下， 还可以做出若干改进和润饰， 这些改进和润饰也应视为本发明的保护范围。

Claims

权 利 要 求
1 、 一种融合蛋白， 包括： （ 1 )特异性结合肿瘤细胞或者肿瘤血管内 皮细胞的靶向结合区； （2 ) 白细胞介素。
2、 如权利要求 1所述的融合蛋白， 其特征在于， 所述融合蛋白还包 含由不多于 10个氨基酸组成的连接区， 用于连接所述靶向结合区和白细 胞介素。
3、 如权利要求 1所述的融合蛋白， 其特征在于， 所述白细胞介素为 人源白细胞介素 2 , 具有如 SEQ ID NO: 1所示的氨基酸序列。
4、 如权利要求 1或 2或 3所述的融合蛋白， 其特征在于， 所述靶向 结合区为不少于 3个但不多于 50个氨基酸的氨基酸序列。
5、 如权利要求 4所述的融合蛋白， 其特征在于， 所述靶向结合区为 人源氨基酸序列。
6、 如权利要求 4所述的融合蛋白， 其特征在于， 所述靶向结合区与 生长抑素同源。
7、 如权利要求 6所述的融合蛋白， 其特征在于， 所述靶向结合区的 氨基酸序列与生长抑素同源性不低于 60%。
8、 如权利要求 6或 7所述的融合蛋白， 其特征在于， 所述靶向结合 区的氨基酸序列为 FCYWKSCT, 如 SEQ ID NO: 2所示， 或为 SEQ ID NO. 1 所示的氨基酸序列经过取代、 缺失、 或添加一个或几个氨基酸衍生的与 SEQ ID NO. 1所示的氨基酸序列至少有 80%的一致性， 且具有特异结合肿 瘤细胞或者肿瘤血管内皮细胞的活性。
9、 如权利要求 8所述的融合蛋白， 其特征在于， 将如 SEQ ID NO: 2 所示的靶向结合区中酪氨酸由任一含苯环或杂环的氨基酸替换，优选替换 为苯丙氨酸或色氨酸。
10、 如权利要求 9所述的融合蛋白， 其特征在于， 将如 SEQ I D NO: 2 所示的靶向结合区中丝氨酸由任一含有羟基的氨基酸替换或缺失，优选替 换为苏氨酸。
11、如权利要求 1-8任一项所述的融合蛋白， 其特征在于， 其中所述 融合蛋白含有如 SEQ ID NO: 3的氨基酸序列。
12、 编码权利要求 1-11任一项所述融合蛋白的 DNA分子。
1 3 、 根据权利要求 12所述的 DNA分子， 其特征在于， 其含有如 SEQ ID NO: 4所示的核苷酸序列。
14、 一种表达盒， 包括： 转录起始区； 受转录起始区调控的编码权 利要求 1-11任一项所述融合蛋白的 DNA分子； 以及转录中止区。
15、 重组了编码权利要求 1-11任一项所述融合蛋白 DNA分子的表达 载体。
16、 如权利要求 15所述的表达载体， 其特征在于， 所述表达载体为 质粒或病毒。
17、 重组了编码权利要求 1-11任一项所述融合蛋白的 DNA分子的细 胞。
18、 如权利要求 17所述的细胞， 其特征在于， 所述细胞为哺乳动物 细胞、 昆虫细胞、 酵母菌或细菌。
19、 根据权利要求 17所述的细胞， 其特征在于， 所述细胞属大肠杆 菌，保藏在中国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. 3867。
20、 一种转基因动物， 其特征在于， 转染了编码权利要求 1-1 1任一 项所述融合蛋白的 DNA分子且表达所述融合蛋白。
21、 权利要求 1-11任一项所述融合蛋白的制备方法， 包括： 提供权利要求 17-19任一项所述细胞或权利要求 20所述转基因动物； 使细胞或者转基因动物表达所述融合蛋白； 以及分离所述融合蛋白。
22、 如权利要求 1-11任一项所述融合蛋白在制造治疗细胞过度增生 导致的疾病的药物中的用途。
23、 如权利要求 22所述的用途， 其特征在于， 所述疾病为癌症。
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