WO2015100634A1 - TNFα与DC-SIGN的融合蛋白及其应用 - Google Patents

TNFα与DC-SIGN的融合蛋白及其应用 Download PDF

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WO2015100634A1
WO2015100634A1 PCT/CN2013/091159 CN2013091159W WO2015100634A1 WO 2015100634 A1 WO2015100634 A1 WO 2015100634A1 CN 2013091159 W CN2013091159 W CN 2013091159W WO 2015100634 A1 WO2015100634 A1 WO 2015100634A1
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fusion protein
tumor
cells
sign
gene
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PCT/CN2013/091159
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French (fr)
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马永
侯景
姚翔
罗成
徐春林
陈晨
王耀方
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江苏众红生物工程创药研究院有限公司
常州京森生物医药研究所有限公司
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Publication of WO2015100634A1 publication Critical patent/WO2015100634A1/zh

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • 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
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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

Definitions

  • the present invention relates to the field of genetic engineering, and relates to a fusion protein of TNFa and DC-SIGN and uses thereof, and more particularly, the present invention relates to a specific intercellular adhesion factor 3 binding non-integration comprising a DC surface which recognizes a variant or a high expression sugar chain.
  • Fusion protein of the extracellular domain of the molecule DC-SIGN, dendritic-cell-specific ICAM-3 -grabbing nonintegrin
  • a linker peptide and tumor necrosis factor (TNF).
  • Targeted anticancer drugs mainly include monoclonal antibodies and small molecule protein kinase inhibitors, depending on the recognition site.
  • the mechanism of action of monoclonal antibodies is to use some specific tumor antigens on the surface of tumor cells as targets for attack, while small molecule protein kinase inhibitors can target abnormal protein kinase receptors, thereby inhibiting tumor growth, differentiation, metabolism and other organisms.
  • the process of learning has an anti-tumor effect.
  • monoclonal antibody drugs and kinase inhibitor drugs have only a single recognition site for tumor cells, and have limited killing effect on cancer cells with mutation or structural changes at the recognition site, so they are mostly combined therapeutic drugs in clinical practice, and their markets are numerous. It is monopolized by multinational companies and the price is very expensive. Therefore, the development of targeted anti-tumor drugs with better targeting and lower efficacy will greatly improve the quality of production of patients with malignant tumors and reduce the cost of medical expenses for patients.
  • Lectins are a class of non-enzymatic, non-antibody proteins that selectively recognize sugars and non-covalently reversibly bind them.
  • the structure of DC-SIGN/CD209 (Dendritic Cell-Specific Intercellular adhesion molecule-3 - Grabbing Non-integrin) contains a calcium-dependent carbohydrate recognition domain (CRD), which belongs to the category of C-type lectin.
  • DC-SIGN can broadly bind to carbohydrate structures derived from pathogens, such as saccharide conjugates containing mannose and Lewis blood group Ags Le x , Le y , Le a and Le b containing fucose ( .
  • DC-SIGN also has the ability to specifically recognize and bind tumor cell sugar markers, including CEA, Lewis blood group antigen, Mac-2BP, Span-1 and the like. This binding is achieved by mutual recognition of the CRD region of the C-type lectin and the tumor-specific sugar chain (Man, D-GlcNA C , Fuc).
  • Apoptosis is a process of energy-dependent cell-ordered active apoptosis controlled by genes.
  • the combination of apoptotic factors and receptors can cause apoptotic pathways, effectively inhibit or kill cells, and can also be used in the development of new anti-tumor drugs.
  • Tumor necrosis factor (TNF) as the most potent cytokine discovered so far, has been clinically studied in Europe and the United States in the 1980s, but it has been forced to terminate due to its extensive biological effects and toxic side effects. Therefore, how to reduce the toxic side effects under the premise of ensuring the therapeutic effect of TNF becomes a key issue for the tumor necrosis factor family members as anti-tumor drugs.
  • the patents CN1458977A and CN1865444A have used a single chain of monoclonal antibodies as a targeting domain for fusion expression with TNFct.
  • the targeting function of such fusion proteins is dependent on the targeting of single-chain antibodies (scFv).
  • scFv single-chain antibodies
  • the single-chain antibody has a complicated structure, poor binding ability, unstable expression, and a single recognition site, and the antigenic site cannot be targeted if it is mutated, thereby affecting the overall function of the monoclonal antibody. Therefore, it is of great significance to develop a stable anti-tumor drug with high specific recognition of tumor cells. Summary of the invention
  • a first object of the present invention is to provide a targeted anti-tumor fusion protein comprising a recognition domain and an action domain, and a linker peptide linking the two domains.
  • the recognition domain is a C-type lectin located at the C-terminus of the fusion protein;
  • the functional domain is a tumor necrosis factor located at the N-terminus of the fusion protein;
  • the linker peptide is a short peptide containing 8-25 amino acids.
  • the identification function mentioned above is DC-SIGN/CD209. More preferably, the recognition function described above is the extracellular region of DC-SIGN/CD209.
  • the above-described functional domain is more preferably TNFc, and the above-described functional domain is the extracellular region of TNFct.
  • the linker peptide described above is a flexible linker peptide.
  • amino acid sequence of the fusion protein described above is shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • Another object of the present invention is to provide a gene expressing the above-described targeted anti-tumor fusion protein, which is preferably represented by SEQ ID NO: 1 or SEQ ID NO: 3.
  • Another object of the present invention is to provide a plasmid comprising the above-described gene.
  • the plasmid is p3FLAG-CMV-13 or pCHO 1.0.
  • Another object of the present invention is to provide a mammalian cell comprising the above-described plasmid.
  • Another object of the present invention is to provide a pharmaceutical preparation or a pharmaceutical composition comprising the fusion protein described above.
  • the pharmaceutical composition may also comprise at least one other therapeutic agent, such as an antibody, a kinase inhibitor or a cancer vaccine or the like.
  • Another object of the present invention is to provide a novel use of the above-described fusion protein in the preparation of an antitumor drug.
  • the new use is for the treatment of colorectal cancer.
  • the present invention connects tumor necrosis factor and C-type lectin through a linker peptide.
  • Such fusion proteins simultaneously utilize the targeted binding of C-type lectin to cancer cells and tumor necrosis factor-mediated apoptosis to induce apoptosis in cancer cells.
  • the combination of targeting function and pro-apoptotic ability allows the fusion protein to specifically kill cancerous cells while reducing the extent of its effects as much as possible, ensuring that normal tissues and cells are not killed.
  • the inventors have unexpectedly discovered that the fusion of C-type lectins also confers the property of protein multimerization.
  • the fusion protein forming a multimer increases the local pro-apoptotic signal, thereby enhancing the cross-reaction of tumor cells and immune cells, and greatly promoting the anti-tumor immunity of the body, so that the overall biological activity of the fusion protein has Great improvement.
  • Figure 1 is a multiple sequence alignment map.
  • FP fusion protein
  • TNFa tumor necrosis factor
  • Linker GGGGGGGGGG linker peptide (G10 for short)
  • DC-SIGN specific intercellular adhesion factor 3 on the surface of DC binds to non-integrin molecule.
  • FIG. 2 is a structural simulation diagram of the TNFa-G10-DC-SIGN fusion protein.
  • G1-L177 TNFa
  • G178-G187 G10 linking peptide
  • Q188-A533 DC-SIGN.
  • FIG. 3 is a graphical comparison of the TNFa structure in the TNFa monomer and the fusion protein TNFa-G10-DC-SIGN.
  • a structural alignment of 33 - 177 amino acids was performed, and the resulting RMSD value was 0.429 A, indicating that the functional domain of TNFa in the fusion protein did not change with the monomeric protein TNFa.
  • the TNFa end of TNFa and AT3002 fusion protein almost completely coincide. Therefore, it can be speculated that the TNFa end of the AT3002 fusion protein is substantially identical to the protein tertiary structure of the TNFa monomer, that is, the TNFa in the AT3002 fusion protein retains the natural biological activity of TNFa.
  • the boxed area of the figure indicates the region where the TNF receptor interacts with TNFa.
  • Figure 4 is a detailed view of the docking of the fusion protein with the receptor, which is a partial enlarged view of the boxed area of Figure 3.
  • the upper part is the fusion protein AT3002 and the lower part is the TNFa receptor. It can be seen that the binding between TNFa and its receptor in the fusion protein AT3002 has no steric hindrance and can interact.
  • Figure 5 shows a comparison of nucleotide sequences before and after codon optimization of the AT3002 fusion protein.
  • the odd rows are the sequences before the AT3002 gene codon optimization
  • the even rows are the codon-optimized sequences of the AT3002 gene.
  • Figure 6-a shows that the nucleotide sequence of the AT3002 gene has a CAI index calculated to be 0.84 in a mammalian cell expression host;
  • Figure 6-b shows the optimized AT3002 gene nucleotide sequence codon of the present invention in a mammal
  • the CAI index in the cell expression host was calculated to be 0.85 by the program.
  • Figure 7 is a diagram showing the optimal codon frequency distribution region in mammalian cell expression hosts before and after AT3002 gene codon optimization.
  • Figure 7-a shows the optimal codon frequency distribution of the nucleotide sequence of the AT3002 gene in mammalian cell expression hosts. It can be seen from the figure: Percentage of low-utilization codons of the nucleotide sequence of the AT3002 gene 5%;
  • Figure 7-b shows the optimal codon frequency distribution region map of the optimized AT3002 gene of the present invention in a mammalian cell expression host, and the optimized low utilization codon of the AT3002 gene codon sequence of the present invention The percentage of occurrence is 1%.
  • Figure 8 is a map of the average GC base content distribution in mammalian cell expression hosts before and after AT3002 gene codon optimization.
  • Figure 8-a shows that the average nucleotide base content of the AT3002 gene nucleotide sequence in the mammalian cell expression host is: 57.25%;
  • Figure 8-b shows the optimized expression of the AT3002 gene codon of the present invention in mammalian cells.
  • the average GC base content in the host is: 54.57%.
  • Figure 9 is a schematic diagram showing the structure of a eukaryotic expression vector.
  • Fig. 9-a is a schematic view showing the structure of the eukaryotic expression vector p3xFLAG-CMV-13.
  • Figure 9-b is a schematic view showing the structure of the eukaryotic expression vector pCHOl.O.
  • Figure 10 is an agarose gel electrophoresis pattern of AT3002 fusion protease digestion (Hind III, BamH I double digestion). Among them, Lane 1 is double-digested with the expression vector p3xFLAG-CMV-13 containing the AT3002 gene; Lane 2 is a 200 bp DNA ladder.
  • Figure 11 is an immunoblot showing the expression of AT3002 in HLF cells.
  • Lane 1 is a pre-stained protein ladder; lanes 2 and 3 are HLF cell lysates and cell culture supernatants; lanes 4 and 5 are HLF/AT3002 cell lysates and cell culture supernatants, respectively.
  • Figure 12 is an immunoblot of CHO cell expression AT3002.
  • Figure 12a is an immunoblot of AT3002 expression after transfer of plasmid p3xFLAG-CMV-13-AT3002 into CHO cells.
  • Lane 1 is a pre-stained protein ladder;
  • Lane 2 is a cell lysate supernatant of HLF cells expressing AT3002 (positive control);
  • Lane 3 is a cell lysate supernatant of CHO cells expressing AT3002.
  • Figure 12b is an immunoblot of AT3002 expression after plasmid pCHO1.0-AT3002 was transferred into CHO cells.
  • Lane 1 is a pre-stained protein ladder;
  • Lane 2 is a CHO original cell culture supernatant (blank control);
  • Lane 3 is a CHO cell-expressing AT3002 cell lysate supernatant.
  • Figure 13 shows the results of in vitro anti-tumor experiments of HLF cells expressing AT3002 fusion protein (MTT method)
  • Figure 13-a shows the results of killing of SW1116 colon cancer cells by HLB cells expressing AT3002 fusion protein
  • Figure 13-b shows the killing effect of AT3002 fusion protein on COLO205 colon cancer cells
  • Figure 13-c shows the AT3002 fusion protein pair
  • Figure 13-d shows the killing test results of AT3002 fusion protein on T84 colon cancer cells
  • Figure 13-e shows the killing test results of AT3002 fusion protein on LoVo colon cancer cells.
  • the experimental results show that AT3002 has a killing effect on the above five colorectal cancer cells, and there is a certain dose-effect relationship.
  • Figure 14 is a multimerization assay (immunoblotted map) of HLB cell expression AT3002.
  • lanes 1 and 3 are pre-stained protein ladders; lanes 2 and 4 are respectively AT3002 fusion proteins under reducing conditions and non-reducing conditions. It is indicated that the AT3002 fusion protein expressed in recombinant cells is mostly in the form of multimer.
  • Figure 15 is a Ca 2+ -dependent binding assay ( ⁇ , immune cell fluorescence) of HLB cells expressing the AT3002 fusion protein to different colon cancer cells.
  • the figure shows the binding of AT3002 to SW1116, COLO205, LS 174T, T84, LoVo cells under different conditions.
  • the results show that AT3002 can specifically bind to the above colon cancer cells, and the binding has Ca 2+ dependence.
  • Figure 16 is a graph showing the results of in vitro anti-tumor experiments of CHO cells expressing AT3002 fusion protein (MTT method).
  • Figure 17 is a Ca 2+ -dependent binding assay ( ⁇ , immune cell fluorescence) of CHO cells expressing AT3002 fusion protein to COLO205 colon cancer cells.
  • Figure 18 is a photomicrograph of HLF/AT3002 cell microcarrier culture. Among them, Fig. 18-a and Fig. 18-b are photomicrographs at 40x and ⁇ , respectively.
  • Figure 19 is an electropherogram or immunoblot of the AT3002 protein before and after purification.
  • Figure 19-a shows the 10x protein concentration of the AT3002 collection supernatant.
  • Lane 1 is a pre-stained protein ladder;
  • Lane 2 is a 10-fold concentrated AT3002 collection supernatant.
  • Figure 19-b shows the electropherogram of the protein after purification of the AT3002 protein.
  • Lane 6 is a pre-stained protein ladder; lanes 1-5 and 7-11 are collections of different collection tubes after AT3002 purification; lane 12 is flow-through.
  • Figure 19-c is an immunoblot of the AT3002 protein after purification.
  • Figure 20 is a graph showing tumor growth inhibition of COLO205 colon cancer by HLF/AT3002 cells.
  • Figure 21 is a graph showing the tumor weight of each group of HLO/AT3002 cells treated with COLO205 colon cancer.
  • Figure 22 is a photograph of a tumor of each group of HLO/AT3002 cells treated with COLO205 colon cancer test.
  • Figure 23 is a photograph of a tumor-bearing mouse in each group of HLO/AT3002 cells treated with COLO205 colon cancer.
  • Figure 24 is a staining diagram of tumor tissue sections of each group of HLO/AT3002 cells treated with COLO205 colon cancer test.
  • Figure 25 is a graph showing tumor growth of HLF/AT3002 cells inhibiting LS 174T colon cancer.
  • Figure 26 is a graph showing the tumor weight of each group of HLF/AT3002 cells treated with the LS 174T colon cancer test.
  • Figure 27 is a graph showing the tumor growth curve of AT3002 fusion protein inhibiting the growth of xenografted tumor in nude mice.
  • Figure 28 is a graph showing the inhibition of growth of transplanted tumors in nude mice by AT3002 fusion protein.
  • Figure 29 shows the effect of AT3002 fusion protein on the survival of nude mice xenografts.
  • Figure 30 is a computer-aided design of the AT3132 fusion protein.
  • Figure 30-a is a structural simulation of the AT3132 fusion protein, G1-L177: TNFa; E178-K192: linker peptide (EAAAK) 3 ; Q193-A538: DC-SIGN.
  • Figure 30-b is a simulated alignment of the TNFa structure in the TNFa monomer and the AT3132 fusion protein, and the boxed region shows the region where the TNFa receptor interacts with TNFa.
  • TNFa in fusion proteins There was no change in the functional domain and the monomeric protein TNFct. It can be seen from the figure that TNFct and the TNFct end of the AT3132 fusion protein are almost completely coincident.
  • FIG. 30-c is a detailed view of the docking of the AT3132 fusion protein with the TNFa receptor. It can be seen that the binding between TNFa and its receptor in the fusion protein AT3132 is sterically hindered and can interact.
  • Figure 31 is an immunoblot of CHO cells expressing the AT3132 fusion protein.
  • Lane 1 is the cell secretory supernatant of the CHO/AT3002 fusion protein;
  • Lane 2 is the pre-stained protein ladder;
  • Lane 3 is the CHO cell culture supernatant that has not been transfected into the gene (negative control group);
  • Lane 4 is CHO/AT3132 Cell culture supernatant.
  • Figure 32-a and Figure 32-b show the results of biological activity assay of the AT3132 fusion protein.
  • Figure 32-a shows the killing test results of AT3132 fusion protein on COLO205 colon cancer cells. The results show that AT3132 and A3002 can effectively kill colorectal cancer cells compared with control TNFa;
  • Figure 32-b is AT3132 fusion protein Ca 2+ -dependent binding assay to COLO205 cells ( ⁇ , immune cell fluorescence). The results showed that AT3132 binds to colorectal cancer cells with Ca 2+ dependence.
  • the present invention first selects the linker peptide G10 as a linker peptide of TNFct and DC-SIGN.
  • the T cell immunogenicity scores of G10 were calculated as: 5 (highest score), 0, 0.
  • the B cell immunogenicity score calculated using BceprecL ABCpred was: 0, 0.51, the normalized T cell and B cell immunogenic scores were 0.0 and 0.51, respectively, which were weakly immunogenic.
  • DC-SIGN is used as the recognition functional domain
  • the number of the GenBank corresponding to DC-SIGN is M98457
  • the corresponding protein number is AAF77072 (Fig. 1).
  • the specific cell of DC-SIGN is called DC surface.
  • Inter-adhesion factor 3 binds to a non-integrin molecule.
  • the amino acid of DC-SIGN used in the present invention is 59-404.
  • TNFa is used as the functional domain in the present invention.
  • the number corresponding to TNFa in GenBank is X01394, and the corresponding protein number is NP_000585 (Fig. 1), which is called tumor necrosis factor alpha.
  • the amino acid used in TNFa of the present invention is 57-233.
  • TNFc G10 and DC-SIGN are ligated in tandem to form a new fusion protein amino acid sequence.
  • the new fusion protein has an amino acid length of 533 amino acids, and the sequence is shown in SEQ ID NO: 2.
  • the fusion protein TNFa-G10-DC-SIGN (or TNFa/DC-SIGN) is collectively referred to as "AT3002" in the present application.
  • DC-SIGN and TNFa use the Template Identification of SWISS-MODEL software to find the template, the template obtained by TNFct (PDB number 1A8M, B chain), and the three-dimensional structure from 82-233 amino acids, need to construct 57-81 amino acids;
  • DC- The three-dimensional structure (PDB number 2IT5) from 253 to 384 amino acids in the SIGN amino acid sequence requires the construction of three-dimensional structures of 59-252 and 384-404 amino acids.
  • the TNFa and DC-SIGN templates were used for modeller modeling after removal of water molecules and other non-covalently bound molecules.
  • the Modeller program was used to construct a homology model of monomeric proteins, including skeletal construction, circular structure simulation, and side chain addition.
  • the DC-SIGN three-dimensional structure model has a total of 346 amino acids, and the TNFa three-dimensional structure model has a total of 177 amino acids.
  • molecular dynamics is applied to optimize the system energy and molecular dynamics.
  • the evaluation method is applied using the Structural Analysis and Verification Server, and the parameters indicated in the evaluation that do not meet the requirements of the stereochemistry are adjusted.
  • the three-dimensional structure model of DC-SIGN and TNFa is spatially rotated and translated so that the longest axes of the two are approximately parallel and the distance is about 20A, and the two three-dimensional structures are written into the same three-dimensional structure file.
  • TNFa belongs to the tumor necrosis factor superfamily.
  • the superfamily has many members, but all have a sequence of about 150 amino acids (TNF homeodomains, THD), which is a conserved backbone containing aromatic and hydrophobic residues.
  • THD in different TNF family protein structures has almost identical spatial folding configurations and is closely related to the formation of trimer proteins.
  • the corresponding receptors of the TNF family are also very numerous, and the characteristic structure is the extracellular domain cysteine-rich domains (CRDs), usually with six cysteine residues involved in the formation of three disulfide bonds.
  • CCDs cysteine-rich domains
  • TNF family protein molecules form trimers, and then their ligands form receptor dimers with receptors. mode.
  • the present invention uses the TNFa and TNFa-R (PDB number 3ALQ) interaction mode as a starting point for molecular docking.
  • TNFa and TNFa-R were performed using RosettaDock online software (http: ⁇ rosettadock.graylab.jhu.edu). Molecular docking of TNFa and TNFa-R in the TNFa-G10-DC-SIGN fusion protein was performed in the same manner (Fig. 3).
  • the three-dimensional structural model of DC-SIGN and TNFa monomers was structurally compared with the DC-SIGN and TNFa domains in the fusion protein using the Superpose program of the CCP4 software package (Fig. 4), considering that DC-SIGN and TNFa have a large number of The ring structure, in the molecular dynamic simulation, will undergo a relatively large change, which will have a great impact on the results of the structural comparison. Therefore, the skeleton structure that maintains its function is selected for structural comparison.
  • the skeletal structure of the 202-322 amino acid of DC-SIGN monomer was structurally aligned with the corresponding part of the fusion protein, and the obtained RMSD value was 0.426A, and the corresponding quantitative parameter (Tm-score) was 0.991.
  • the body was selected for 33-177 amino acids for structural alignment.
  • the obtained RMSD value was 0.429A, the corresponding quantitative parameter (Tm-score) was 0.988, and the two average values were 0.9895.
  • the docking results of the two monomers and the fusion protein DC-SIGN and TNFa were compared, and the molecular docking results of the pre-fusion receptor and the ligand and the molecular docking result of the receptor and the ligand after fusion were compared, and the TNFa was calculated.
  • the structure change, the RMSD value is 0.555A, and the corresponding quantization parameter (Tm-score) is 0.976.
  • the distance between the TNFa and the ligand in the fusion protein is between 2.9 and 3.9 A, which can form a more stable hydrogen bond.
  • the applicant also performed codon optimization, mRNA structure modification and optimization of translation initiation sites.
  • the gene sequence was obtained as shown in SEQ ID NO: 1.
  • the gene comparison before and after optimization is shown in Figure 1.
  • CAI Codon Adaptation Index
  • the codon adaptation index (CAI) of the AT3002 gene in mammalian cells was 0.84 before the codon was optimized.
  • the CA300 index of the AT3002 gene of the present invention in mammalian cells was 0.85 after codon optimization.
  • CAI codon adaptation index
  • the gene is considered to be the most ideal high-efficiency expression state in the expression system.
  • the lower the CAI index indicates that the expression level of the gene in the host is worse, so it can be seen that after the codon optimization
  • the gene sequence can increase the expression level of the AT3002 gene in mammalian cells. 2.
  • Frequency of Optimal Codons (FOP) Frequency of Optimal Codons
  • the percentage of low utilization codons of the AT3002 gene sequence was 5% before the codon was optimized.
  • This unoptimized gene contains tandem rare codons that can reduce translation efficiency and even disintegrate translational assemblies.
  • the AT3002 gene of the present invention has a low utilization codon frequency of 1% in the mammalian cell system after codon optimization.
  • the ideal distribution of GC content is 30%-70%, and any peak outside this region will affect the conversion and translation efficiency to varying degrees. From the comparison of the GC base average content distribution area maps of the AT3002 gene in Fig. 8-a and Fig. 8-b, it can be seen from Fig. 8-a that the average content of GC bases in the AT3002 gene before optimization is 57.25%, from Fig. 8 The -b showed that the average GC base of the heavy AT3002 gene was 54.57%, which was more favorable for the expression of the AT3002 gene.
  • the optimized AT3002 fusion protein gene was artificially synthesized, and Hind III and BamH I restriction sites were added to both ends of the fusion protein gene. A stop codon site was introduced at the same time after the BamH I site to prevent the expression of the FLAG tag on the expression vector from affecting the properties of the fusion protein.
  • the fusion protein gene was inserted into the p3xFLAG-CMV-13 plasmid (Fig. 9a, plasmid purchased from Sigma) by the above two restriction enzyme sites to obtain a long-term preservation plasmid, which was designated as p3xFLAG-CMV-13-AT3002 plasmid. .
  • the optimized AT3002 fusion protein gene was artificially synthesized, and the restriction sites of Avr II and BstZ 171 were added to the entire length of the fusion protein gene.
  • the fusion protein gene was inserted into the pCHO 1.0 plasmid (Fig. 9b, plasmid purchased from Invitrogen) through the above two restriction enzyme sites to obtain a long-term preservation plasmid, which was designated as pCHO. 1.0-AT3002 plasmid.
  • the eukaryotic expression vector p3xFLAG-CMV-13-AT3002 synthesized above was transferred into Escherichia coli DH5a (purchased from Tiangen Biochemical Technology Co., Ltd.), cultured and amplified, and the plasmid was extracted.
  • the plasmid was subjected to restriction enzyme digestion (Fig. 10), and the result of restriction enzyme digestion showed that the gene size of the above AT3002 was as expected.
  • the amplified plasmid was transferred to HLF cells (purchased from JCRB cell bank) by electroporation.
  • the specific operations are as follows:
  • HLF cells are cultured to a concentration of 60%-80%, trypsin (purchased from Amersco), and then resuspended in the original culture solution;
  • Electrotransformation conditions The electrorotation voltage is 110V, the electrosurgical time is 25ms, and the cell culture solution is added immediately after the electric shock;
  • ASF104 serum-free medium purchased from Ajinomoto, Japan
  • serum-free medium was collected and the medium was concentrated.
  • the cells were collected with a spatula and resuspended in 500 uL of universal lysis buffer (ration: 25 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP40, 5% Glycine, pH 7.4 o before adding 1% protease inhibitor) in 4 °C was cleaved for 1 h.
  • the primary antibody used for immunoblotting was a mouse anti-human DC-SIGN antibody (purchased from R&D) and the secondary antibody was HRP-rabbit anti-mouse antibody (purchased from Sigma).
  • the theoretical molecular weight of AT3002 is 60 kd, and the concentrated medium and cell lysate in step 2 are separated by 10% SDS-PAGE gel.
  • the specific bands were detected in the intracellular and extracellular products of HLF/AT3002, while the expression of AT3002 was not detected in the HLF empty cell control of the control lanes 2 and 3.
  • the intracellular AT3002 and the secreted AT3002 have a molecular weight of about 10 kd.
  • the secreted AT3002 has a larger molecular weight than intracellular, which may be related to the post-translational modification of the fusion protein.
  • the plasmids p3xFLAG-CMV-13-AT3002 and pCHO1.0-AT3002 in Example 3 were separately transferred into CHO cells (purchased from ATCC) by the method of Example 4, and the expression was cultured, and expression was identified by immunoblotting. As shown in Figure 12, the AT3002 fusion protein was also expressed in CHO cells. CHO is an internationally recognized recombinant protein and therapeutic monoclonal antibody expressing cell line. The expression of AT3002 in CHO cells also suggests that the AT3002 fusion protein possesses the potential of industrialization.
  • Example 6 Detection of Biological Activity of HLF Cell Expressing AT3002 Fusion Protein
  • colon cancer cell line COLO205 purchased from JCRB cell bank
  • SW1116 purchased from JCRB cell bank
  • LS 174T purchased from Shanghai Library of Chinese Academy of Sciences
  • T84 purchased from Shanghai Institute of Chinese Academy of Sciences
  • Library and LoVo (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) to study the in vitro antitumor activity of the fusion protein.
  • the antitumor activity was measured by the MTT method.
  • the supernatant of the HLF/AT3002 fusion protein-expressing cell line cultured in ASF104 serum-free medium purchasedd from Ajinomoto, Japan was cryopreserved and used for MTT assay.
  • the above colorectal cancer cells were cultured in a 24-well plate at a concentration of 50,000 cells/well while adding the fusion protein culture supernatant or TNFct standard (purchased from PraSpec, Israel) to be detected while adding 10 mM Ca 2+ . After 48 h, MTT (final concentration of 500 ug/mL) was added, incubation was continued for 4 h, the culture was terminated, and the culture supernatant in the well was carefully aspirated. For the suspension cells, it is necessary to centrifuge and then aspirate the culture supernatant in the wells. Add 400 uL of DMSO to each well and shake for 10 min to allow the crystals to fully melt.
  • MTT final concentration of 500 ug/mL
  • the wavelength of 490 nm was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor. The larger the absorbance value, the stronger the cell viability, and the smaller the absorbance value, the more the tumor cells were inhibited or killed.
  • Figures 13-a through 13-e are graphs showing the killing test results of the AT3002 fusion protein on these five colon cancer cells, respectively.
  • the AT3002 has a certain killing effect on SW1116, COLO205, LS 174T, ⁇ 84 and LoVo.
  • the AT3002 has a dose-dependent effect on COLO205, LS 174T and T84.
  • Figure 14 is a multimerization analysis of the AT3002 fusion protein. It can be seen from the figure that the molecular weight of the non-denatured fusion protein is much larger than that of the denatured fusion protein, which indicates that the fusion protein is multimerized, resulting in an increase in molecular weight.
  • the formation of multimers is one of the characteristics of C-type lectins, and the multimerization of fusion proteins is likely due to the multimerization of C-type lectins.
  • the multimerized fusion protein is capable of locally enhancing the pro-apoptotic signal and increasing its anti-tumor activity.
  • This experiment was carried out by the method of cellular immunofluorescence experiment. Briefly as follows: 24-well or 96-well plate cell slides (COLO205, SW1116); aspirate the culture supernatant, rinse with TBS 3 times; 4% PFA (purchased from Sigma) for 20 min, TBS wash three times; Serum against the same host (or 5% skim milk) was blocked for 1 h, TBS was washed three times; the treated cells were incubated with the fusion protein secretion supernatant for 1 h (adding 10 mM Ca 2+ , 10 mM EDTA or 10 mM mannose), primary antibody ( Mouse anti-human DC-SIGN antibody, purchased from R&D, diluted with 2.5% skim milk) overnight at 4 ° C, or lh at room temperature, washed three times with TBS; Fluorescently labeled secondary antibody (Alexa-514 rabbit anti-mouse labeled antibody, diluted with TBS) at room temperature for 1 h (protected from
  • Figure 15 is an immunofluorescence micrograph of AT3002 and colon cancer cells.
  • the binding of the lectin to the sugar chain is Ca 2+ dependent.
  • AT3002 can bind to these colon cancer cells and has a strong fluorescent signal.
  • AT3002 not only has good antitumor activity, but also has a sugar chain-specific binding ability.
  • Example 7 CHO Cell Expression Biological Activity Detection of AT3002 Fusion Protein
  • FIG. 16 shows the killing ability of AT3002 expressed by CHO on COLO205 cells. As can be seen from the figure, the cell viability decreased significantly in the AT3002 group relative to the control group, indicating that the AT3002 fusion protein expressed by CHO cells also has good tumor cell killing activity.
  • HLF cells are adherent cells and cannot be cultured in suspension.
  • the present invention performs expanded culture of HLF cells by a method of suspension culture of microcarriers.
  • Cytodexl microcarriers were purchased from GE. A suitable amount of microcarriers was dissolved in PBS and sterilized. MEM medium (purchased from Qingda Tianyi Bio) was added to a sterilized shake flask, and 3-5 g/L of microcarriers were added, and the amount of cells inoculated was 100,000 cells/ mL, after 3 days of culture, replace ASF104 serum-free medium. The supernatant was collected after 2-3 days of incubation for fusion protein Separation and purification.
  • Figure 18-a, Figure 18-b are photomicrographs of microcarrier cultured HLF/AT3002 cells. As shown, HLF cells are able to grow normally on microcarriers. This finds a suitable method for HLF/AT3002 expansion culture (suspension culture).
  • HLF/AT3002 expansion culture suspension culture.
  • the AT3002 fusion protein was purified by affinity chromatography with mannan-agarose filler. Sample pretreatment:
  • the sample is loaded at a flow rate of 1 -4 mL/min;
  • Elution buffer Proportion: lOmM Tris, lOOmM NaCl, 10 mM EDTA, pH 7.4
  • Protein SDS-PAGE electrophoresis and immunoblotting were used to detect protein purity.
  • Figure 19 is a diagram showing the protein electrophoresis of AT3002 before and after purification. It can be seen from the pictures that there is no impurity band of the purified AT3002 fusion protein, and the purified AT3002 has no impurity bands, indicating that the purification effect is better.
  • Example 10 Cellular therapeutic effect of HLF cells expressing fusion protein AT3002 on COLO205 tumors
  • the invention selects a nude mouse xenograft model to investigate the antitumor activity of the fusion protein.
  • the inhibitory effect of HLF cells secreting fusion protein on the growth of colon cancer COLO205 cell xenografts was investigated by inoculating HLF cells expressing the fusion protein with colon cancer cells COLO205.
  • the specific experimental protocol is as follows: The cells in vitro were resuspended at a concentration of 10 8 /mL, and the experiment was divided into model group (COLO205), AT3002 group (COLO205+HLF/AT3002) and two control groups (COLO205+HLF/DC-SIGN). ; COLO205+HLF/X-DC-SIGN ), 9-10 per group.
  • the cells inoculated with COLO205+HLF/DC-SIGN control group only expressed the targeting domain domain DC-SIGN; the cells inoculated with COLO205+HLF/X-DC-SIGN control group expressed the targeting domain domain DC-SIGN and Unrelated protein (human serum albumin) fusion-expressed HLF cells.
  • HLF/DC-SIGN and HLF/X-DC-SIGN cell lines were prepared according to the method of Example 4, and the target gene DC-SIGN or X-DC-SIGN was inserted into the p3xFLAG-CMV-13 vector and electrotransformed. HLF cells, positive clone cell lines were obtained.
  • the cells were mixed in equal proportions according to the grouping, and female nude mice of 6-8 weeks old were injected subcutaneously to construct a nude mouse xenograft model.
  • Nude mice were purchased from the Shanghai Slack Animal Center and were inoculated with tumors after 5 days of adaptive feeding. After the inoculation, the growth of the tumor was monitored, and the size of the tumor was calculated according to the length X width X width/2. three weeks After the nude mice were sacrificed, the tumor was removed and the tumor weight was weighed. In addition, paraffin sections and immunochemical tissue staining were performed on tumor tissues to detect the expression of AT3002 in tumor tissues. Tissue embedding and paraffin sectioning were completed at Zhenjiang First People's Hospital.
  • the staining of tumor tissue sections was performed according to the conventional staining method (diagnostic immunohistochemistry, 2011 edition, edited by Ji Xiaolong, P14-P39), the primary antibody for incubation was DC-SIGN antibody (purchased from R&D), and the secondary antibody was HRP- Rabbit anti-mouse antibody (purchased from Sigma). Only the chromogenic substrate took a different TMB chromogenic solution (purchased from AMRESCO) than the reference method.
  • Figure 20 is a graph showing the growth curves of each group of cell treatments.
  • tumor growth of nude mice inoculated with HLF/AT3002 was significantly inhibited, and the presence of tumor was hardly detected in the later stage.
  • the tumor growth of the model group and the two control groups was very significant.
  • the same results were obtained for the tumor weight of nude mice (Fig. 21).
  • Fig. 22 and Fig. 23 are photographs of tumors of each group, and also reflect the growth of tumors of each group.
  • Fig. 22 is a photograph of the exfoliated tumor
  • Fig. 23 is a photograph of the tumor-bearing mouse before the tumor is exfoliated.
  • the tumors of the model group and the control group were larger than those of the AT3002 group, indicating that the tumor growth of the AT3002 group was inhibited.
  • This experiment also stained sections of tumor tissue (Fig. 24). The staining results showed that the color reaction of tumor tissue in the AT3002 group showed that AT3002 was expressed and enriched in the tumor; while the control group COLO205 did not develop color. This suggests that AT3002 specifically binds to tumor tissue and inhibits the growth of tumor cells.
  • Example 11 Expression of fusion protein HLF cells for cell therapy of LS 174T tumors
  • Example 10 The anti-LS 174T colon cancer effect of HLF/AT3002 was studied by the method of Example 10.
  • the specific experimental scheme was the same as that of Example 10, and was divided into two groups: negative control group, HLF+COLO205; AT3002 group, HLF/AT3002+ COLO205.
  • Figure 25 is a graph showing the growth curves of the two groups of tumors. It can be seen that the tumor growth rate of the AT3002 group was significantly inhibited compared with the control group.
  • Figure 26 shows the same results for the weight of the two groups of tumors.
  • Example 12 Evaluation of in vivo antitumor activity of AT3002 fusion protein
  • the present invention selects a nude mouse xenograft model to investigate the antitumor activity of AT3002.
  • a nude mouse xenograft model was constructed by subcutaneous injection of 6-8 week old female nude mice with COLO205 cells. The purified AT3002 fusion protein was intraperitoneally injected into nude mice to observe the tumor growth. The specific operations are as follows: Nude mice were purchased from Shanghai Slack Animal Center. After 5 days of adaptive feeding, COLO205 cells cultured in vitro were resuspended at a concentration of 10 8 /mL, and each nude mouse was subcutaneously inoculated with 10 7 COLO205 cells (200 uL). .
  • the results of the experiment showed that the growth of transplanted tumors in both the high and low dose groups was inhibited compared with the control group (Fig. 27, Fig. 28).
  • the tumor inhibition rates of the low dose group and the high dose group were 45.0% and 57.7%, respectively. It can be seen that the inhibition rate shows a significant dose-effect relationship with the dose administered.
  • the experiment also counted the survival time of tumor-bearing mice. The statistical results showed (Fig. 29). After 50 days, the nude mice in the control group were basically dead. Compared with the control group, the survival time of the AT3002 protein-administered group was Significant improvement. It can be seen that AT3002 can significantly inhibit and kill tumor cells, and can effectively prolong the survival of tumor-bearing mice.
  • Example 13 Design and Preliminary Activity Evaluation of AT3132 Fusion Protein
  • the present invention also selects a linker peptide which also belongs to a molecule having good molecular flexibility: (EAAAK) 3 , which is capable of forming a helical high-order structure and sufficiently stretching the protein domain linked to both ends thereof.
  • the present invention also studied (EAAAK) 3 as a fusion protein of TNFct and DC-SIGN linker peptides.
  • EAAAK linker peptide which also belongs to a molecule having good molecular flexibility:
  • the present invention also studied (EAAAK) 3 as a fusion protein of TNFct and DC-SIGN linker peptides.
  • the fusion protein TNFa-(EAAAK) 3 -DC-SIGN is collectively referred to as "AT3132" in the present application, and the amino acid sequence thereof is shown in SEQ ID NO: 4.
  • the present invention also further simulates the advanced structure of AT3132 by the method of Embodiment 1 (Fig. 30-a).
  • the structural alignment of the simulated AT3132 fusion protein with the monomeric TNFa protein shows that the N-terminus of AT3132 is almost completely coincident with TNFct, indicating that the structural changes of the two are small.
  • the molecular docking simulation test shows that the docking of AT3132 with TNFaR is consistent with its monomeric protein, which further demonstrates that the structure of the AT3132 fusion protein does not affect the activity of its TNFa domain.
  • FIG. 31 is a diagram showing the immunoblotting of the AT3132 protein. As can be seen from the figure, the molecular size of AT3132 is similar to that of AT3002; the brightness of the band is also the same as that of A3002, indicating that the expression levels of the two are also similar.
  • the present invention investigated the killing ability and binding ability of AT3132 to COLO205 cells by the method of Example 6. The results (FIG. 32-a, FIG.
  • AT3132 inhibit the growth of colon cancer cell COLO205; AT3132 also has the ability to bind Ca 2+ dependency, which can be EDTA, and the ability to bind mannose (mannose) inhibition.
  • the experimental results of the present invention indicate that TNFa using the (EAAAK) 3 linker peptide has better biological activity as the DC-SIGN fusion protein.

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Abstract

本发明公开了TNFα与DC-SIGN的融合蛋白及其应用，该融合蛋白由识别功能域和作用功能域，以及连接这两个功能域的连接肽组成。所述识别功能域是具有肿瘤细胞表面糖链识别及结合功能的凝集素多肽--DC表面的特异性细胞间黏附因子3结合非整合素分子（DC-SIGN），位于融合蛋白的C端；所述作用功能域是肿瘤坏死因子（TNF），位于融合蛋白的N端；所述连接肽为含有8-25个氨基酸的短肽。该融合蛋白可以利用凝集素与癌细胞进行靶向结合，通过促细胞凋亡配体诱导癌细胞凋亡的同时降低了对正常组织和细胞的杀伤作用。

Description

TNFa与 DC-SIGN的融合蛋白及其应用 技术领域
本发明涉及基因工程领域， 涉及 TNFa与 DC-SIGN的融合蛋白及其应用， 更具体而言， 本发明涉及包含识别变异或高表达糖链的 DC表面的特异性细胞间黏附因子 3结合非整合素 分子 （DC-SIGN, dendritic-cell-specific ICAM-3 -grabbing nonintegrin) 的胞外区结构域、 连接 肽和肿瘤坏死因子 （TNF ) 的融合蛋白。 背景技术
随着人们生活环境及生活方式的改变， 抗肿瘤市场正在逐步扩大， 目前治疗肿瘤的放疗 和化疗等常规手段无法区分肿瘤细胞和正常组织细胞， 常常导致严重的副作用， 往往在杀伤 肿瘤细胞的同时也破坏机体正常的免疫功能， 使患者的生存、 生活质量大大下降。 因此减少 现在的肿瘤治疗药物的毒副作用成为亟需解决的课题， 目前研究已表明靶向性抗肿瘤药物具 有高效、 低毒副作用和低成本肿瘤治疗的特点， 已成为近期研究的热点。
根据识别位点的不同， 靶向抗癌药物主要包括单克隆抗体和小分子蛋白激酶抑制剂。 单 抗的作用机理是利用肿瘤细胞表面有一些特异的肿瘤抗原可作为攻击靶点， 而小分子蛋白激 酶抑制剂能靶向抑制异常蛋白激酶受体， 从而抑制肿瘤的生长、 分化、 代谢等生物学过程而 起到抗肿瘤的疗效。 然而， 单抗药物和激酶抑制剂药物对肿瘤细胞仅有单一的识别位点， 对 识别位点有突变或结构改变的癌症细胞杀伤效果有限， 因此临床上多为联合治疗药物， 且其 市场多被跨国公司所垄断， 价格非常昂贵。 因此， 开发靶向性和疗效更好、 价格更为低廉的 靶向抗肿瘤药物将大大提高恶性肿瘤患者的生产质量， 降低患者的医疗开销费用。
凝集素 (lectin) 是一类能选择性地识别糖并与之非共价可逆结合的非酶、 非抗体蛋白质。 DC-SIGN/CD209 ( Dendritic Cell-Specific Intercellular adhesion molecule-3 -Grabbing Non-integrin)的结构中含有钙依赖型的糖类识别区域（carbohydrate recognition domain, CRD ) , 属于 C型凝集素的范畴。 DC-SIGN可以广泛结合病原体来源的糖类结构， 如包含有甘露糖的 糖结合物以及包含岩藻糖的路易斯血型抗原（Lew blood group Ags) Lex、 Ley、 Lea和 Leb ( . Nonaka et al" J Biol Chem 286, 22403 ) DC-SIGN还具有特异性识别和结合肿瘤细胞糖标志物 的能力， 这些肿瘤糖标志物包括 CEA、 Lewis血型抗原、 Mac-2BP、 Span-1等。 这种结合通 过 C型凝集素的 CRD区域和肿瘤特异性糖链 （Man，D-GlcNAC， Fuc) 相互识别来实现。
细胞凋亡 (Apoptosis)是由基因控制的能量依赖性细胞有序的主动凋亡的过程。 细胞凋亡 因子与受体结合后可引起细胞凋亡途径，有效抑制或杀伤细胞，亦可用于抗肿瘤新药的研发。 肿瘤坏死因子（TNF)作为迄今发现的抗肿瘤活性最强的细胞因子， 早在上世纪 80年代欧美 曾对其展开临床研究， 但由于其生物效应广泛、 毒副作用较大而被迫终止。 因此， 如何在保 证 TNF治疗效应的前提下降低其毒副作用成为肿瘤坏死因子家族成员能否作为抗肿瘤药物的 关键性问题。 专利 CN1458977A和 CN1865444A曾采用单克隆抗体的单链作为靶向结构域与 TNFct融合表达。 这类融合蛋白的靶向功能依赖于单链抗体（scFv) 的靶向性。 但是， 单链抗 体结构复杂、 结合能力差、 表达不稳定， 且识别位点单一， 抗原位点如发生突变等即无法达 到靶向的作用，从而影响到了单抗的整体功能。因此，开发一种具有肿瘤细胞高特异识别的、 稳定的抗肿瘤药物具有重要的意义。 发明内容
为克服上述不足， 本发明第一个目的是提供一种靶向抗肿瘤融合蛋白， 其由识别功能域 和作用功能域， 以及连接这两个功能域的连接肽组成。 所述识别功能域是 C型凝集素， 位于 融合蛋白的 C端； 所述作用功能域是肿瘤坏死因子， 位于融合蛋白的 N端； 所述连接肽为含 有 8-25个氨基酸的短肽。
优选的， 上述所述的识别功能域是 DC-SIGN/CD209。 更优选的， 上述所述的识别功能域 是 DC-SIGN/CD209的胞外区。
优选的， 上述所述的作用功能域是 TNFc 更优选的， 上述所述的作用功能域是 TNFct 的胞外区。
优选的， 上述所述的连接肽是柔性连接肽。
优选的，上述所述的连接肽的氨基酸序列为 GGGGGGGGGG i¾(EAAAK)n i¾(GGGGS)n, 其中 n=3，4或 5。
优选的， 上述所述的融合蛋白的氨基酸序列如 SEQ ID NO: 2或 SEQ ID NO: 4所示。 本发明的另一个目的是提供一种表达上述所述靶向抗肿瘤融合蛋白的基因， 所述基因优 选如 SEQ ID NO: 1或 SEQ ID NO: 3所示。
本发明的另一个目的是提供一种含有上述所述基因的质粒。 优选的， 所述质粒为 p3FLAG-CMV-13或 pCHO 1.0。
本发明的另一个目的是提供一种含有上述所述质粒的哺乳动物细胞。
本发明的另一目的是提供一种包含上述所述的融合蛋白的药物制剂或药物组合物。 所述 药物组合物还可以包含至少一种其他治疗剂， 如抗体、 激酶抑制剂或癌症疫苗等。
本发明的另一目的是提供上述所述的融合蛋白在制备抗肿瘤药物中的新用途。 优选的， 所述新用途为治疗大肠癌。 本发明将肿瘤坏死因子和 C型凝集素通过连接肽相连。这类融合蛋白同时利用 C型凝集 素与癌细胞的靶向结合以及肿瘤坏死因子介导的细胞凋亡作用诱导癌细胞凋亡。 靶向功能和 促凋亡能力的结合使得该类融合蛋白在特异性杀伤癌变细胞的同时， 尽可能的降低了其作用 的广泛性， 确保正常组织和细胞不被杀死。 除此之外， 发明人还意外发现 C型凝集素的融合 还赋予了蛋白多聚化的性质。 形成多聚体的融合蛋白增加了局部的促凋亡信号， 从而加强了 肿瘤细胞和免疫细胞的交叉反应， 很大程度上促进了机体的抗肿瘤免疫抗力， 使得融合蛋白 整体的生物活性有了很大的提高。 附图说明
图 1为多序列比对图。 FP: 融合蛋白； TNFa: 肿瘤坏死因子； Linker: GGGGGGGGGG 连接肽 （简称 G10) ； DC-SIGN: DC表面的特异性细胞间黏附因子 3结合非整合素分子。
图 2为 TNFa-G10-DC-SIGN融合蛋白的结构模拟图。 G1-L177: TNFa; G178-G187: G10 连接肽； Q188-A533: DC-SIGN。
图 3为 TNFa单体与融合蛋白 TNFa-G10-DC-SIGN中的 TNFa结构模拟比对。 选择 33 — 177氨基酸进行结构比对， 所得 RMSD值为 0.429A, 表明融合蛋白中的 TNFa的功能域与 单体蛋白 TNFa没有什么变化。从图中可以看出 TNFa与 AT3002融合蛋白的 TNFa端几乎完 全重合。因此， 可以推测 AT3002融合蛋白中的 TNFa端与 TNFa单体的蛋白三级结构基本一 致， 即 AT3002融合蛋白中的 TNFa仍然保留了 TNFa的天然生物学活性。 图示方框区域表示 TNF 受体与 TNFa相互作用区域。
图 4为融合蛋白与受体对接细节图， 是图 3的方框区域的局部放大图。 其中上半部分为 融合蛋白 AT3002, 下半部分为 TNFa受体。 可见融合蛋白 AT3002中的 TNFa与其受体间的 结合无空间位阻， 可以发生相互作用。
图 5表示 AT3002融合蛋白密码子优化前后核苷酸序列比较。
其中，奇数行（即"原始序列"对应的行）为 AT3002基因密码子优化前的序列；偶数行（即 "优化序列"对应的行） 为 AT3002基因密码子优化后的序列。
图 6 AT3002基因密码子优化前后在哺乳动物细胞表达宿主中 CAI 指数。
其中， 图 6-a表示 AT3002基因核苷酸序列在哺乳动物细胞表达宿主中 CAI 指数经过程 序计算为 0.84 ； 图 6-b表示优化后的本发明的 AT3002基因核苷酸序列密码子在哺乳动物细 胞表达宿主中 CAI 指数经过程序计算为 0.85。
图 7为 AT3002基因密码子优化前后在哺乳动物细胞表达宿主中最优密码子频率分布区 域图。 其中， 图 7-a表示 AT3002基因核苷酸序列在哺乳动物细胞表达宿主中最优密码子频率 分布区域图，从图中可以看出： AT3002基因核苷酸序列的低利用率密码子出现百分比为 5% ； 图 7-b 表示优化后的本发明的 AT3002基因在哺乳动物细胞表达宿主中最优密码子频率分布 区域图， 优化后的本发明的 AT3002基因密码子序列的低利用率密码子出现百分比为 1%。
图 8为 AT3002基因密码子优化前后在哺乳动物细胞表达宿主中平均 GC碱基含量分布区 域图。
其中，图 8-a表示 AT3002基因核苷酸序列在哺乳动物细胞表达宿主中平均 GC碱基含量 为： 57.25% ； 图 8-b 表示优化后的本发明的 AT3002基因密码子在哺乳动物细胞表达宿主中 平均 GC 碱基含量为： 54.57%。
图 9为真核表达载体结构示意图。
其中， 图 9-a为真核表达载体 p3xFLAG-CMV-13的结构示意图。 图 9-b为真核表达载体 pCHOl.O的结构示意图。
图 10为 AT3002融合蛋白酶切鉴定 （Hind III， BamH I双酶切） 的琼脂糖凝胶电泳图。 其中，泳道 1为含 AT3002基因的表达载体 p3xFLAG-CMV-13双酶切后；泳道 2为 200bp DNA ladder。
图 11为 HLF细胞表达 AT3002的免疫印迹图。 其中， 泳道 1为预染蛋白 ladder; 泳道 2 和 3分别是 HLF的细胞裂解液和细胞培养上清； 泳道 4和 5分别是 HLF/AT3002的细胞裂解 液和细胞培养上清。
图 12为 CHO细胞表达 AT3002的免疫印迹图。
图 12a为质粒 p3xFLAG-CMV-13-AT3002转入 CHO细胞后 AT3002表达的免疫印迹图。 其中， 泳道 1为预染蛋白 ladder; 泳道 2是 HLF细胞表达 AT3002的细胞裂解液上清 （阳性 对照）； 泳道 3是 CHO细胞表达 AT3002的细胞裂解液上清。
图 12b为质粒 pCHO1.0-AT3002转入 CHO细胞后 AT3002表达的免疫印迹图。 其中， 泳 道 1为预染蛋白 ladder; 泳道 2是 CHO原始细胞培养上清（空白对照）； 泳道 3是 CHO细胞 表达 AT3002的细胞裂解液上清。
图 13为 HLF细胞表达 AT3002融合蛋白的体外抗肿瘤实验结果 （MTT法）
图 13-a为 HLF细胞表达 AT3002融合蛋白对 SW1116结肠癌细胞的杀伤性实验结果； 图 13-b为 AT3002融合蛋白对 COLO205结肠癌细胞的杀伤性实验结果；图 13-c为 AT3002融合 蛋白对 LS 174T结肠癌细胞的杀伤性实验结果； 图 13-d为 AT3002融合蛋白对 T84结肠癌细 胞的杀伤性实验结果； 图 13-e为 AT3002融合蛋白对 LoVo结肠癌细胞的杀伤性实验结果。 实验结果表明， AT3002对上述五株大肠癌细胞均具有杀伤作用， 且存在一定的量效关系。 图 14为 HLF细胞表达 AT3002的多聚化分析（免疫印迹图）。 其中， 泳道 1、 3是预染蛋 白 ladder; 泳道 2、 4分别是还原条件和非还原条件下 AT3002融合蛋白。 说明重组细胞中表 达的 AT3002融合蛋白多以多聚体的形式存在。
图 15为 HLF细胞表达 AT3002融合蛋白与不同结肠癌细胞的 Ca2+依赖性结合实验（ ΙΟΟχ, 免疫细胞荧光）。 图为 AT3002与 SW1116 、 COLO205、 LS 174T、 T84、 LoVo细胞在不同条 件下的结合图， 结果显示 AT3002可与上述结肠癌细胞特异结合， 且结合具有 Ca2+依赖性。
图 16为 CHO细胞表达 AT3002融合蛋白的体外抗肿瘤实验结果图 （MTT法）。
图 17为 CHO细胞表达 AT3002融合蛋白与 COLO205结肠癌细胞的 Ca2+依赖性结合实验 ( ΙΟΟχ, 免疫细胞荧光）。
图 18为 HLF/AT3002细胞微载体培养的显微镜照片。 其中， 图 18-a和图 18-b分别是在 40x和 ΙΟΟχ倍下的显微照片。
图 19为 AT3002蛋白纯化前后的电泳图或免疫印迹图。
图 19-a为 AT3002收集上清浓縮 10倍的蛋白电泳图。 其中， 泳道 1为预染蛋白 ladder; 泳道 2为 10倍浓縮的 AT3002收集上清。图 19-b为 AT3002蛋白纯化后的蛋白电泳图。其中， 泳道 6为预染蛋白 ladder; 泳道 1-5和 7-11为 AT3002纯化后收集后不同收集管的收集液； 泳 道 12为流穿液。图 19-c为 AT3002蛋白纯化后的免疫印迹图。其中，泳道 6为预染蛋白 ladder; 泳道 1-5和 7-11为 AT3002纯化后收集后不同收集管的收集液； 泳道 12为流穿液。
图 20为 HLF/AT3002细胞抑制 COLO205结肠癌的肿瘤生长曲线图。
图 21为 HLF/AT3002细胞治疗 COLO205结肠癌试验各组的肿瘤重量统计图。
图 22为 HLF/AT3002细胞治疗 COLO205结肠癌试验各组的肿瘤照片。
图 23为 HLF/AT3002细胞治疗 COLO205结肠癌试验各组的荷瘤鼠照片。
图 24为 HLF/AT3002细胞治疗 COLO205结肠癌试验各组的肿瘤组织切片染色图。
图 25为 HLF/AT3002细胞抑制 LS 174T结肠癌的肿瘤生长曲线图。
图 26为 HLF/AT3002细胞治疗 LS 174T结肠癌试验各组的肿瘤重量统计图。
图 27为 AT3002融合蛋白抑制裸鼠移植瘤生长的肿瘤生长曲线图。
图 28为 AT3002融合蛋白抑制裸鼠移植瘤生长的统计图。
图 29为 AT3002融合蛋白对裸鼠移植瘤小鼠生存期的影响。
图 30为 AT3132融合蛋白的计算机辅助设计图。
其中， 图 30-a为 AT3132融合蛋白的结构模拟图， G1-L177: TNFa; E178-K192: 连接肽 (EAAAK)3 ; Q193-A538 : DC-SIGN。 图 30-b为 TNFa单体与 AT3132融合蛋白中的 TNFa结 构模拟比对， 图示方框区域表示 TNFa受体与 TNFa相互作用区域。 融合蛋白中的 TNFa的 功能域与单体蛋白 TNFct没有什么变化。 从图中可以看出 TNFct与 AT3132融合蛋白的 TNFct 端几乎完全重合。因此， 也可以推测 AT3132融合蛋白中的 TNFct端与 TNFct单体的蛋白三级 结构仍然保持一致， 也就是说可以推测 AT3132融合蛋白中的 TNFct端可以仍然保留 TNFa 单体的生物学活性。 图 30-c为 AT3132融合蛋白与 TNFa受体对接细节图， 可见融合蛋白 AT3132中的 TNFa与其受体间的结合无空间位阻， 可以发生相互作用。
图 31为 CHO细胞表达 AT3132融合蛋白的免疫印迹图。 其中， 泳道 1为 CHO/AT3002 融合蛋白的细胞分泌上清； 泳道 2为预染蛋白 ladder; 泳道 3是未转入基因的 CHO细胞培养 上清 （阴性对照组）； 泳道 4是 CHO/AT3132的细胞培养上清。
图 32-a与图 32-b为 AT3132融合蛋白的生物学活性检测结果。 其中， 图 32-a为 AT3132 融合蛋白对 COLO205结肠癌细胞的杀伤性试验结果，结果显示，与对照 TNFa相比， AT3132 与 A3002—样可以有效杀伤大肠癌细胞；图 32-b为 AT3132融合蛋白与 COLO205细胞的 Ca2+ 依赖性结合实验 （ΙΟΟχ, 免疫细胞荧光）。 结果显示， AT3132与大肠癌细胞结合具有 Ca2+依 赖性。 具体实施方式
现在结合以下实施例说明本发明。 提供这些实施例仅用于说明的目的， 本发明不限于这 些实施例， 而是包含明显由本文提供的教导产生的所有改变。 实施例 1融合蛋白的基因设计
一、 连接肽筛选
本发明首先选取连接肽 G10作为 TNFct与 DC-SIGN的连接肽。
应用 SYFPEITHI、 MH2pred、 A Npred 软件， 并分别采用其默认参数计算， 算得 G10 的 T细胞免疫原性分数为： 5 (最高分）、 0、 0， 应用 BceprecL ABCpred计算 B细胞免疫原 性分数为： 0、 0.51， 分别归一化后计算的 T细胞和 B细胞免疫原性分数为 0.0和 0.51， 都属 于弱免疫原性。
二、 蛋白模型构建
本发明优选采用 DC-SIGN作为识别功能域， DC-SIGN所对应的 GenBank中的编号为 M98457, 所对应的蛋白编号为 AAF77072 (；图 1 )， DC-SIGN的全称为 DC表面的特异性细胞 间黏附因子 3结合非整合素分子。本发明中所用的 DC-SIGN的氨基酸为 59-404。本发明优选 采用 TNFa作为作用功能域， TNFa所对应的 GenBank中的编号为 X01394, 所对应的蛋白编 号为 NP_000585(图 1 )，全称为肿瘤坏死因子 alpha。本发明中的 TNFa所用的氨基酸为 57-233。 按顺序将 TNFc G10和 DC-SIGN的氨基酸 （以单字母表示） 序列串联， 形成新的融合 蛋白氨基酸序列，新的融合蛋白氨基酸长度为 533个氨基酸，该序列如 SEQ ID NO: 2所示。 为表述方便， 本申请中统一将该融合蛋白 TNFa-G10-DC-SIGN (或 TNFa/DC-SIGN ) 记作 "AT3002
DC-SIGN和 TNFa分别应用 SWISS-MODEL软件的 Template Identification寻找模板， TNFct得到的模板（PDB编号 1A8M， B链）， 从 82-233氨基酸都已经有三维结构， 需要构建 57-81氨基酸； DC-SIGN氨基酸序列中从 253至 384氨基酸已有三维结构（PDB编号 2IT5 ), 需要构建 59-252和 384-404氨基酸三维结构。 TNFa和 DC-SIGN模板去除水分子和其他非共 价结合的分子后用于 modeller建模。
采用 Modeller程序进行单体蛋白同源模型构建， 包括骨架构建、 环状结构模拟以及侧链 添加。 DC-SIGN三维结构模型共有 346个氨基酸， TNFa三维结构模型共有 177个氨基酸。初 始模型构建完成后， 应用分子动力学进行系统能量最优化和分子动力模拟， 所用 Sander程序 的主要参数为： Maxcyc=2500，Ncyc=1000， Cutoff=10。 分子动力学模拟完成之后应用一些评 价程序（Structural Analysis and Verification Server)进行模型评估， 对于评估中指出的不符合立 体化学要求的参数进行调整。
将 DC-SIGN和 TNFa的三维结构模型在空间上进行旋转和平移，使两者的最长轴近似平 行且距离约为 20A， 将这两个三维结构写入同一个三维结构文件， 以此做为新的模板应用 Modeller程序来构建融合蛋白三维结构模型 （图 2)。 融合蛋白模型构建完成后应用分子动力 学进行系统能量最优化和分子动力模拟， 所用 Sander 程序的主要参数为： Maxcyc=2500,Ncyc=1000, Cutoff=10。 对融合蛋白的模型进行评估， 对于评估中指出的不符 合立体化学要求的参数进行调整。
三、 分子对接
TNFa属于肿瘤坏死因子超家族的成员， 该超家族成员众多， 但都有一段长约 150个氨 基酸的序列， （TNF homeodomains, THD)，该序列是含有芳香族和疏水性残基的保守骨架。 不 同 TNF家族蛋白结构中的 THD有着几乎完全相同的空间折叠构型， 并且与三聚体的蛋白形 成有密切关系。 TNF家族的对应的受体也非常多， 特征性结构是其胞外域富含半胱氨酸的结 构域（CRDs), 通常有 6个半胱氨酸残基参与形成三个二硫键。从已有的研究成果来看， TNF 家族的受体与配体的相互作用模式是多种多样的，但是首先是 TNF家族蛋白分子形成三聚体， 然后其配体与受体形成二聚体模式。 在 TNFP 与 TNFP-R (PDB 编号 1TNR)、 TRAIL 与 TRAIL-R2 (PDB编号 1D4V)、 TNFa与 TNFa-R(PDB编号 3 ALQ)都是以 3:3结合模式产出 相互作用。 本发明以 TNFa与 TNFa-R(PDB编号 3ALQ)相互作用模式作为分子对接的起点。 采用 RosettaDock在线软件 （http：〃 rosettadock.graylab.jhu.edu) 对 TNFa禾卩 TNFa-R进行分子 对接。 应用同样的方式对 TNFa-G10-DC-SIGN融合蛋白中的 TNFa和 TNFa-R进行了分子对 接 （图 3 )。
四、 结构比对与打分
应用 CCP4软件包的 Superpose程序对 DC-SIGN和 TNFa单体三维结构模型与融合蛋白 中的 DC-SIGN和 TNFa结构域进行结构比对 （图 4)， 考虑到 DC-SIGN和 TNFa中具有大量 的环形结构， 在分子动力模拟中会发生比较大的变化， 会对结构比对结果产生很大的影响， 所以选择维护其功能的骨架结构进行结构比对。对 DC-SIGN单体选择 202-322氨基酸的骨架 结构与融合蛋白中相对应的部分进行结构比对，所得 RMSD值为 0.426A,相对应的量化参数 (Tm-score) 为 0.991 ; 对 TNFa单体选择 33— 177氨基酸进行结构比对， 所得 RMSD值为 0.429A, 相对应的量化参数 （Tm-score) 为 0.988， 两平均值为 0.9895。 两个单体与融合蛋白 中 DC-SIGN和 TNFa由获得的分子对接结果，比较了融合前受体与配体的分子对接结果和融 合后受体与配体的分子对接结果， 计算了 TNFa的结构变化， RMSD值为 0.555A, 相对应的 量化参数 （Tm-score) 为 0.976。 此外， 融合蛋白中的 TNFa与其配体相互作用的残基距离在 2.9-3.9A之间， 可以形成较稳定的氢键。 由此可见， 根据计算机的模拟结果可以得出融合蛋 白中的识别功能域 DC-SIGN和作用功能域 TNFa的空间结构与天然状态下各单体的空间结构 没有明显的差异， 并且融合蛋白中的 TNFa依然保存了其生物活性， 可以高效的与其受体结 合并发生相互作用。 实施例 2 AT3002融合蛋白的基因优化
1. 密码子优化
为了能更好的表达该 AT3002融合蛋白， 申请人还对其进行了密码子优化， mRNA结构 修正以及翻译起始位点的优化。 得到基因序列如 SEQ ID NO: 1所示。 优化前后的基因对比 如图 1所示。
下面是对 AT3002基因进行密码子优化前后各参数对比如下：
1. 密码子适应指数 （Codon Adaptation Index, CAI)
由图 6-a可知，密码子没有优化前， AT3002基因在哺乳动物细胞中密码子适应指数 (CAI) 为 0.84。 由图 6-b可知， 通过密码子优化后， 使得本发明的 AT3002基因在哺乳动物细胞中 CAI 指数为 0.85。 通常 CAI=1 时被认为该基因在该表达系统中是最理想的高效表达状态， CAI 指数越低表明该基因在该宿主中表达水平越差， 因此可以看出经过了密码子优化后得到 的基因序列可以提高 AT3002基因在哺乳动物细胞中的表达水平。 2. 最优密码子使用频率 （Frequency of Optimal Codons, FOP)
由图 7-a可知， 基于哺乳动物细胞表达载体， 密码子没有优化前， AT3002基因序列的低 利用率密码子出现百分比为 5%。这条未进行优化的基因含有串联稀有密码子，这些密码子可 能降低翻译效率， 甚至能够解散翻译装配物。 由图 7-b 可知， 通过密码子优化后， 本发明的 AT3002基因在哺乳动物细胞系统中出现低利用率密码子的频率为 1%。
3. GC 碱基含量 （GC curve)
GC 含量理想分布区域为 30%-70%， 在这个区域外的出现任何峰都会不同程度地影响转 录和翻译效率。 由图 8-a、 图 8-b 的 AT3002基因的 GC 碱基平均含量分布区域图对比可知， 由图 8-a 中显示 AT3002基因中在优化前 GC碱基平均含量为 57.25%， 由图 8-b中显示最终 得到优化后重 AT3002基因的 GC 碱基平均含量为 54.57%， 更有利于 AT3002基因的表达。
4. 优化前后顺式作用元件情况如下：
Figure imgf000011_0001
5.优化前后回文以及重复序列情况如下:
Figure imgf000011_0002
实施例 3 AT3002融合蛋白的基因合成和表达载体构建
将上述优化后的 AT3002融合蛋白全基因进行人工合成， 并在融合蛋白基因全长的两端 分别加入了 Hind III和 BamH I的酶切位点。 BamH I位点后面同时引入了终止密码子位点， 以防止表达载体上 FLAG标签的表达影响融合蛋白的性质。 融合蛋白基因通过上述两个限制 性酶切位点插入到 p3xFLAG-CMV-13 质粒 （图 9a， 质粒购自 Sigma公司） 中， 得到一种长 期保存质粒， 记为 p3xFLAG-CMV-13-AT3002质粒。
将上述优化后的 AT3002融合蛋白全基因进行人工合成， 并在融合蛋白基因全长的两端 分别加入了 Avr II和 BstZ 171的酶切位点。融合蛋白基因通过上述两个限制性酶切位点插入 到 pCHO 1.0质粒（图 9b，质粒购自 Invitrogen公司）中，得到一种长期保存质粒，记为 pCHO 1.0-AT3002质粒。 实施例 4 AT3002融合蛋白在 HLF细胞中的表达和鉴定
一、 重组表达载体电转化 HLF细胞
将上述合成得到的真核表达载体 p3xFLAG-CMV-13-AT3002转入大肠杆菌 DH5a (购自 天根生化科技有限公司）， 培养扩增， 抽提质粒。 对质粒进行酶切验证 （图 10)， 酶切验证结 果显示，上述 AT3002的基因大小与预期一致。将扩增后的质粒用电转化法转入 HLF细胞（购 自 JCRB cell bank) 具体操作如下：
1. 将 HLF细胞培养至 60%-80%的汇集度， 胰酶 （购自 Amersco) 消化后， 重悬于原培 养液中；
2. 1000 rpm, 室温离心 5-7min， 弃上清， 用 2 mL PBS缓冲液重悬；
3. 电转化条件： 电转电压 110V, 电转时间 25ms， 电击后立即加入细胞培养液；
4. 12h后更换为含 1% G418 (购自 Sigma) 的筛选细胞培养液。
二、 阳性克隆蛋白样品的制备
当电转化后的细胞长至铺满培养瓶 80-100%浓度时， 更换 ASF104无血清培养基 （购自 Ajinomoto, Japan)。 2-3d后，收集无血清培养基，浓縮培养基。细胞用刮刀收集后重悬到 500uL 的通用裂解缓冲液（配比： 25mM Tris， 150mM NaCl， 2mM EDTA, 1% NP40, 5% Glycine, pH 7.4 o 用前加入 1%蛋白酶抑制剂） 中在 4°C裂解 lh。
三、 免疫印迹鉴定融合蛋白的表达情况
免疫印迹法检测所用的一抗为鼠抗人 DC-SIGN抗体 （购自 R&D)， 二抗为 HRP-兔抗鼠 抗体 （购自 Sigma)。 AT3002的理论分子量是 60kd， 将步骤二中的浓縮后的培养基及细胞裂 解液 10%的 SDS-PAGE胶分离。 从图 11可以看出， HLF/AT3002的胞内和胞外产物检测到了 特异性的条带， 而对照组 2、 3泳道的 HLF空细胞对照没有检测到 AT3002的表达。 胞内的 AT3002和分泌表达的 AT3002分子量有 10kd左右的差异。分泌的 AT3002分子量比胞内的要 大， 这可能与融合蛋白的翻译后修饰加工有关。 实施例 5 AT3002融合蛋白在 CHO细胞中的表达和鉴定
采用实施例 4的方法将实施例 3中的质粒 p3xFLAG-CMV-13-AT3002和 pCHO1.0-AT3002 分别转入 CHO细胞（购自 ATCC)并培养表达， 用免疫印迹法进行表达鉴定。如图 12所示， AT3002融合蛋白在 CHO细胞内也能够表达。 CHO是国际上通用的重组蛋白和治疗性单克隆 抗体表达细胞株。 AT3002在 CHO细胞内的表达也暗示了 AT3002融合蛋白具备产业化的潜 实施例 6 HLF细胞表达 AT3002融合蛋白的生物学活性检测
一、 融合蛋白的大肠癌细胞杀伤试验
本实验选取糖链特异性高表达的结肠癌细胞系 COLO205 (购自 JCRB cell bank)、 SW1116 (购自 JCRB cell bank)、 LS 174T (购自中科院上海细胞库）、 T84 (购自中科院上海细胞库） 和 LoVo (购自中科院上海细胞库）来研究融合蛋白的体外抗肿瘤活性。 抗肿瘤活性检测采用 MTT法。 用 ASF104无血清培养基 （购自 Ajinomoto, Japan) 培养的 HLF/AT3002融合蛋白 表达细胞株表达上清， 冻存， 备 MTT实验用。 上述大肠癌细胞按照 50000个 /孔的浓度在 24 孔板上培养， 同时加入待检测的融合蛋白培养上清或 TNFct标准品 （购自 PraSpec, Israel), 同时加入 10mM的 Ca2+。 48h后，加入 MTT (终浓度为 500ug/mL)，继续孵育 4h，终止培养， 小心吸弃孔内培养上清液。 对于悬浮细胞需要离心后再吸弃孔内培养上清液。 每孔加 400uL DMSO, 振荡 lOmin, 使结晶物充分融解。 选择 490nm波长， 在酶联免疫监测仪上测定各孔 光吸收值， 吸光值越大说明细胞活力越强， 吸光值越小肿瘤细胞被抑制或被杀死越多。
图 13-a到 13-e分别是 AT3002融合蛋白对这五株结肠癌细胞的杀伤性试验结果图。 由图 可以看出， AT3002对 SW1116、 COLO205、 LS 174T、 Τ84和 LoVo都有一定的杀伤效果。 其 中， AT3002对 COLO205、 LS 174T和 T84的杀伤效果呈剂量依赖性。 这些实验结果表明， AT3002融合蛋白依然保留了 TNFct的肿瘤细胞杀伤能力。
二、 融合蛋白的多聚体形成分析
融合蛋白的多聚化分析用非变性蛋白电泳检测，操作步骤见（《精编蛋白质科学实验指南》 2010版， J.E.科林根等著， P303-P307)。 图 14为 AT3002融合蛋白的多聚化分析。 从图中可 以看出， 非变性的融合蛋白分子量远大于变性的融合蛋白， 这说明融合蛋白发生了多聚化， 导致分子量增加。 而形成多聚体是 C型凝集素的特性之一， 融合蛋白的多聚化很可能是 C型 凝集素的多聚化所致。 多聚化的融合蛋白能够在局部增强促肿瘤凋亡信号， 增加其抗肿瘤活 性。
三、 融合蛋白的糖链结合能力检测
本实验用细胞免疫荧光实验的方法进行检测。 简述如下： 24孔板或 96孔板细胞爬片 (COLO205、 SW1116);吸掉培养基上清， TBS漂洗 3遍； 4%PFA (购自 Sigma)固定 20min， TBS洗三遍； 与二抗相同宿主的血清 （或 5%脱脂牛奶） 封闭 lh， TBS洗三遍； 处理好的细 胞与融合蛋白分泌上清孵育 lh (加入 10mM Ca2+、 10mM EDTA或 10mM 甘露糖）， 一抗（鼠 抗人 DC-SIGN抗体，购自 R&D，用 2.5%脱脂牛奶稀释） 4°C过夜， 或室温 lh， TBS洗三遍； 荧光标记二抗（Alexa-514兔抗鼠标记抗体，用 TBS稀释）室温 lh (避光），或者 37°C45min， PBS洗三遍 （避光）； 封片， 荧光显微镜下观察。
完整有活性的融合蛋白除了能够杀伤肿瘤细胞，还应具有能够靶向结合肿瘤细胞的能力。 本通过细胞免疫荧光实验， 考察融合蛋白与结肠癌细胞 SW1116、 COLO205、 LS 174T、 Τ84 和 LoVo的结合能力。 图 15是 AT3002与结肠癌细胞的免疫荧光显微照片。 凝集素与糖链的 结合具有 Ca2+依赖性。 从图中可以看出， 在加入了 10mM Ca2+后， AT3002能够与这几株结肠 癌细胞结合，有较强的荧光信号。但是加入 10mM EDTA后则抑制了这种结合，这是因为 EDTA 能够螯合 Ca2+， 从而阻止了 C型凝集素与大肠癌细胞的结合。 同时， 加入 50mM的甘露糖 (mannose)能够竞争性地结合融合蛋白与大肠癌细胞结合位点， 导致融合蛋白无法与大肠癌 细胞结合。 另外， 在未加入 10mM Ca2+的对照组也能看到有强的荧光结合， 这说明生理浓度 的 Ca2+也能够满足 AT3002与大肠癌细胞结合的需要。
靶向性是融合蛋白功能性评价中最重要的一环。 只有融合蛋白具有靶向性才能避免正常 组织和细胞免受促凋亡分子的影响， 才能最大程度降低促凋亡蛋白的副作用， 也是药学评价 中最为关注的问题之一。在本实施例的融合蛋白大肠癌细胞杀伤实验中， AT3002不仅有较好 的抗肿瘤活性， 同时也拥有糖链特异性的结合能力。 实施例 7 CHO细胞表达 AT3002融合蛋白的生物学活性检测
采用实施例 6的方法对实施例 5中 CHO表达的 AT3002融合蛋白进行活性检测。 图 16 检验了 CHO表达的 AT3002对 COLO205细胞的杀伤能力。 由图可以看出， AT3002组相对于 对照组， 细胞活力下降很多， 这说明 CHO细胞表达的 AT3002融合蛋白同样具有较好的肿瘤 细胞杀伤活性。
对 CHO细胞表达 AT3002的肿瘤细胞结合活性研究也可以看出 （图 17)， 在加入了 Ca2+ 和 EDTA后， 抑制了 AT3002蛋白与 COLO205细胞的结合能力。 同时， 通过对比 HLF表达 AT3002融合蛋白的结合能力， 可以看出 CHO表达的 AT3002具有更加清晰的荧光显色。 这 说明 CHO表达的 AT3002与肿瘤细胞的结合具有更高的特异性，且结合作用具有 Ca2+依赖性。 实施例 8 HLF/AT3002融合蛋白表达细胞株的扩大培养
HLF细胞是贴壁细胞，无法进行悬浮培养。本发明通过微载体悬浮培养的方法进行了 HLF 细胞的扩大培养。 Cytodexl微载体购自 GE公司。 取适量微载体溶于 PBS并灭菌， 向灭菌的 摇瓶中加入 MEM培养基（购自清大天一生物），加入 3-5g/L的微载体，细胞接种量为 100000 个细胞 /mL， 培养 3d后换 ASF104无血清培养基。 继续培养 2-3d后收集上清， 用于融合蛋白 的分离纯化。 图 18-a、 图 18-b为微载体培养 HLF/AT3002细胞的显微照片。 如图所示， HLF 细胞能够在微载体上正常生长。这为 HLF/AT3002扩大培养（悬浮培养）找到了合适的方法。 实施例 9 AT3002融合蛋白的分离纯化
AT3002融合蛋白用甘露聚糖-琼脂糖填料亲和层析纯化。 样品预处理：
1、 2.6L上清加入 10x 结合缓冲液（配比： 10mM Tris, lOOmM NaCl, lOmM CaCl2, pH 7.4 ) 至总体积为 3L， 混匀；
2、 12000rpm离心 10min， 除去细胞和细胞碎片；
3、 0.45um滤膜过滤。
具体实验步骤：
1、 均匀混合填料， 在 lx结合缓冲液中过夜吸附；
2、 用大于 10个柱体积的结合缓冲液平衡层析柱；
3、 样品按照 1 -4mL/min流速上样；
4、 洗脱： 洗脱缓冲液 (配比： lOmM Tris, lOOmM NaCl, 10mM EDTA，PH 7.4)， 收集洗脱 液。 蛋白 SDS-PAGE电泳和免疫印迹法检测蛋白纯度。
图 19是纯化前后的 AT3002的蛋白电泳图， 由图片可见， 没有纯化的 AT3002融合蛋白 杂带多， 而纯化后的 AT3002基本没有杂带， 说明纯化效果较好。 实施例 10表达融合蛋白 AT3002的 HLF细胞对 COLO205肿瘤的细胞治疗作用
本发明选取裸鼠移植瘤模型来考察融合蛋白的抗肿瘤活性。 通过将表达融合蛋白的 HLF 细胞与结肠癌细胞 COLO205混合接种到裸鼠皮下， 来考察 HLF细胞分泌融合蛋白对结肠癌 COLO205细胞移植瘤生长的抑制作用。 具体实验方案如下： 将体外培养细胞按 108/mL浓度 重悬细胞， 实验分为模型组 （COLO205 )、 AT3002组 （COLO205+HLF/AT3002)和两个对照 组 （ COLO205+HLF/DC-SIGN； COLO205+HLF/X-DC-SIGN )， 每组 9-10 只。 其中， COLO205+HLF/DC-SIGN 对照组接种的细胞仅表达靶向端结构域 DC-SIGN； COLO205+HLF/X-DC-SIGN对照组接种的细胞为表达靶向端结构域 DC-SIGN与无关蛋白（人 血清白蛋白）融合表达的 HLF细胞。 HLF/DC-SIGN和 HLF/X-DC-SIGN细胞株均按照实施例 4的方法进行制备， 将目的基因 DC-SIGN或 X-DC-SIGN插入到 p3xFLAG-CMV-13载体中， 并电转化 HLF细胞， 得到阳性克隆细胞株。 按照分组将细胞等比例混合均匀， 皮下注射 6-8 周龄的雌性裸鼠， 构建裸鼠移植瘤模型。裸鼠购自上海斯莱克动物中心， 适应性伺养 5d后接 种肿瘤。 接种完后， 监测肿瘤的生长情况， 肿瘤的大小按照长 X宽 X宽 /2来计算。 三个星期 后， 裸鼠被处死， 剥离肿瘤， 称量肿瘤重量。 此外， 本实验还对肿瘤组织进行了石蜡切片和 免疫化学组织染色， 来检测肿瘤组织处 AT3002 的表达。 组织包埋和石蜡切片在镇江第一人 民医院完成。 肿瘤组织切片的染色参考常规的染色方法 （《诊断免疫组织化学》 2011 版， 纪 小龙编著， P14-P39), 孵育用的一抗为 DC-SIGN抗体 （购自 R&D)， 二抗为 HRP-兔抗鼠抗 体 （购自 Sigma)。 仅显色底物采取了与参考方法不同的 TMB显色液 （购自 AMRESCO)。
图 20是细胞治疗各组的生长曲线图。 从图中可以看出， 接种了 HLF/AT3002的给药组的 裸鼠肿瘤生长得到了明显的抑制， 到后期几乎检测不到肿瘤的存在。 而模型组和两个对照组 的肿瘤生长非常明显。 对裸鼠肿瘤重量的统计也得到了同样结果 （图 21 )。 图 22和图 23是 各组的肿瘤照片， 也反映了各组肿瘤生长的情况。 图 22是剥离肿瘤的照片， 图 23是肿瘤剥 离前的荷瘤鼠照片。 从图中可以看出， 模型组和对照组的肿瘤比 AT3002组体积要大， 这说 明 AT3002组的肿瘤生长得到了抑制。本实验还对肿瘤组织的切片进行了染色（图 24)。 染色 结果显示， AT3002组的肿瘤组织部位能发生显色反应， 表明 AT3002在肿瘤出表达并富集； 而对照组 COLO205却没有显色。这说明的确是 AT3002可特异性的与肿瘤组织结合并抑制肿 瘤细胞的生长。 实施例 11表达融合蛋白 HLF细胞对 LS 174T肿瘤的细胞治疗作用
采用实施例 10的方法对 HLF/AT3002的抗 LS 174T结肠癌作用进行了研究。具体实验方 案跟实施例 10相同， 分为两组： 阴性对照组， HLF+COLO205; AT3002组， HLF/AT3002+ COLO205。 图 25是两组肿瘤的生长曲线图， 可以看出 AT3002组的肿瘤生长速度相比对照组 受到了明显的抑制。 图 26对两组肿瘤的重量进行了统计也得到了同样结果。 实施例 12 AT3002融合蛋白的体内抗肿瘤活性评价
本发明选取裸鼠移植瘤模型来考察 AT3002 的抗肿瘤活性。 用 COLO205细胞皮下注射 6-8周龄的雌性裸鼠， 构建裸鼠移植瘤模型， 将纯化后的 AT3002融合蛋白腹腔注射裸鼠， 观 察肿瘤生长情况。 具体操作如下： 裸鼠购自上海斯莱克动物中心， 适应性伺养 5d后， 将体外 培养的 COLO205细胞按 108/mL浓度重悬，每只裸鼠皮下接种 107个 COLO205细胞 (200uL)。 7d后， 待肿瘤长至直径 6-7mm时， 剔除少部分未成瘤的裸鼠， 随机分三组 （n=8或 9)。 实 验分为对照组 （TBS-Ca缓冲液）， 小剂量组 （40ug/kg)， 大剂量组 （400ug/kg)三组。 分组后 的小鼠每天腹腔注射给药， 连续给药 10d。 每天监测肿瘤的生长情况， 并统计其生存期。 肿 瘤的大小按照长 X宽 X宽 /2 来计算。 肿瘤生长率计算公式： （肿瘤终末体积-肿瘤初始体积） X 100% /肿瘤初始体积。 肿瘤抑制率计算公式： （对照组肿瘤终末体积一给药组肿瘤终末体 积） X 100% /对照组肿瘤终末体积。
实验结果显示，与对照组相比，高、低剂量组移植瘤的生长都受到了抑制（图 27、图 28)， 小剂量组和大剂量组的肿瘤抑制率分别达到了 45.0%和 57.7%， 可见抑制率与给药剂量呈现 出明显的量效关系。 同时， 本实验还对荷瘤鼠的生存期进行了统计， 统计结果显示（图 29)， 50天后对照组的裸鼠基本都已死亡， 相比对照组， AT3002 蛋白给药组的生存期有了显著的 提高。 由此可见， AT3002可以明显抑制并杀伤肿瘤细胞， 并可以有效延长荷瘤鼠的生存期。 实施例 13 AT3132融合蛋白的设计和初步活性评估
本发明另外也选取了同样属于具有较好分子柔性的连接肽： （EAAAK)3， 它能够形成螺旋 状的高级结构， 使与其两端连接的蛋白结构域充分舒展开来。本发明同样也研究了 (EAAAK)3 作为 TNFct 和 DC-SIGN 连接肽的融合蛋白。 为表述方便， 本申请中统一将融合蛋白 TNFa-(EAAAK)3 -DC-SIGN记作" AT3132", 其氨基酸序列如 SEQ ID NO: 4所示。
本发明也进一步通过实施例 1 的方法模拟出 AT3132 的高级结构 （图 30-a)。 将模拟的 AT3132融合蛋白与单体的 TNFa蛋白进行结构比对 （图 30-b)， 可以看出 AT3132的 N端与 TNFct几乎完全重合， 说明两者的结构变化微小。 分子对接的模拟试验 （图 30-b， 图 30-c) 表明 AT3132与 TNFaR的对接与其单体蛋白的对接一致， 由此可以进一步证明 AT3132融合 蛋白的结构不影响其 TNFa结构域的活性。
本发明按照实施例 2的方法对融合蛋白的基因进行了优化 （优化后的核苷酸序列如 SEQ ID NO: 3所示）， 并将优化后的 AT3132基因按照实施例 5的方法转入 CHO细胞进行表达 和鉴定。 图 31是 AT3132蛋白的免疫印迹法鉴定图。 从图中可以看出， AT3132的分子大小 与 AT3002相近； 条带的亮度也与 A3002相同， 说明两者的表达量也相差不大。 为了验证 AT3132的生物学活性，本发明采取实施例 6的方法研究了 AT3132对 COLO205细胞的杀伤 能力和结合能力。试验结果（图 32-a， 图 32-b)表明 AT3132能够抑制结肠癌细胞 COLO205 的生长； AT3132也同样具有 Ca2+依赖性的结合能力， 这种结合能力能够被 EDTA和甘露糖 (mannose) 抑制。 本发明的实验结果表明采用 (EAAAK)3连接肽的 TNFa与 DC-SIGN融合 蛋白同样具有较好的生物学活性。

Claims

权利要求书
1、 一种靶向抗肿瘤融合蛋白， 其特征在于由识别功能域和作用功能域， 以及连接这两个功能 域的连接肽组成， 所述的识别功能域为 C型凝集素， 位于融合蛋白的 C端； 所述的作用功能 域为肿瘤坏死因子， 位于融合蛋白的 N端； 所述连接肽为含有 8-25个氨基酸的短肽。
2、 根据权利要求 1所述的靶向抗肿瘤融合蛋白， 其特征在于所述的识别功能域为 DC-SIGN, 优选 DC-SIGN的胞外区。
3、 根据权利要求 1所述的靶向抗肿瘤融合蛋白， 其特征在于所述的作用功能域为 TNFct, 优 选 TNFa的胞外区。
4、 根据权利要求 1所述的靶向抗肿瘤融合蛋白， 其特征在于所述的连接肽为柔性、 弹性连接 肽，优选氨基酸序列为 GGGGGGGGGG或 (ΕΑΑΑΚ)η或 (GGGGS)n的连接肽，其中 n=3，4或 5。
5、 根据权利要求 1〜4中任一项所述的靶向抗肿瘤融合蛋白， 其特征在于所述的靶向抗肿瘤融 合蛋白的氨基酸序列如 SEQ ID NO: 2或 SEQ ID NO: 4所示。
6、 编码权利要求 1〜4中任一项所述的靶向抗肿瘤融合蛋白的基因。
7、 根据权利要求 6所述的基因， 其特征在于序列如 SEQ ID NO: 1或 SEQ ID NO: 3所示。
8、含有权利要求 6或 7所述的基因的表达载体，所述表达载体优选以 p3FLAG-CMV-13或 pCHO 1.0为出发质粒。
9、 含有权利要求 6或 7所述的基因的哺乳动物细胞， 所述的哺乳动物细胞优选为 CHO细胞 或 HLF细胞。
10、 一种包含权利要求 1〜4中任一项所述的融合蛋白的药物制剂或药物组合物。
11、 根据权利要求 10所述的药物制剂或药物组合物， 其特征在于所述药物组合物还包含至少 一种其他治疗剂， 所述的其他治疗剂选自抗体、 激酶抑制剂或癌症疫苗。
12、 权利要求 1所述的融合蛋白在制备抗肿瘤药物中的应用。
13、 根据权利要求 12所述的应用， 其特征在于所述的肿瘤为大肠肿瘤。
PCT/CN2013/091159 2013-12-30 2013-12-31 TNFα与DC-SIGN的融合蛋白及其应用 WO2015100634A1 (zh)

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