WO2017036026A1 - 基因修饰的间充质干细胞及其用于生产BsAb抗体的方法 - Google Patents

基因修饰的间充质干细胞及其用于生产BsAb抗体的方法 Download PDF

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WO2017036026A1
WO2017036026A1 PCT/CN2015/099177 CN2015099177W WO2017036026A1 WO 2017036026 A1 WO2017036026 A1 WO 2017036026A1 CN 2015099177 W CN2015099177 W CN 2015099177W WO 2017036026 A1 WO2017036026 A1 WO 2017036026A1
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mesenchymal stem
anticd3
stem cells
genetically modified
antibody
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French (fr)
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王志勇
陈志英
谭燕
杨磊
何成宜
钟育健
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中国科学院深圳先进技术研究院
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • the present invention relates to a genetically modified mesenchymal stem cell and a method thereof for producing a BsAb antibody, and in particular, the present invention relates to a genetically modified mesenchymal stem cell, a preparation method thereof, and a gene-modified mesenchymal stem cell A method of producing a bispecific single chain antibody.
  • Cytotoxic T Lymphocyte CTL
  • Cytokine induced killer play an important role. They possess T cell receptor (TCR) and can The cells expressing the antigen of interest specifically recognize and bind, and secrete perforin and granzyme, thereby killing the target cells of the pathogen.
  • BsAb bispecific antibody
  • the bispecific antibody (BsAb) constructed according to the gene recombination method is capable of simultaneously binding to T cells and cancer cell-specific antigens, and activates the T cell immune mechanism to achieve "dual-targeted antibody-mediated T cell re-reactivation.
  • Anticancer effectors of BsAb-Retargeting T cell This BsAb antibody assists functional T cells in repositioning lesion targets and restoring autoimmune function.
  • the BsAb antibody used is mainly produced by in vitro genetic engineering, and after being purified by the protein, it is intravenously injected from the human body, and finally the BsAb antibody can be delivered to the target region only by blood circulation.
  • the BsAb antibody obtained by this method is extremely easy to inactivate under temperature changes, and the treatment method is expensive; at the same time, the BsAb antibody has a short half-life, thereby reducing the probability of contact with the target cells, so that the desired therapeutic effect cannot be formed.
  • stem cells have been reported.
  • most of the genetic modification methods for stem cells use viral vectors such as lentivirus, adenovirus, and retrovirus.
  • viral vector transfection can improve the efficiency of stem cell genetic modification, due to the safety of the virus, this method has certain limitations, such as strict requirements on the operating environment.
  • the viral gene may be randomly integrated into the cell genome after infection. Therefore, the cell genome is destroyed, causing cancer risk.
  • the transfection efficiency of the viral vector applied to the animal is not high, because the body itself initiates a mechanism for silencing the virus function.
  • RNA ribonucleic acids
  • the main object of the present invention is to use a mesenchymal stem cell to create a biologically active carrier, to provide a genetically modified mesenchymal stem cell, a preparation method thereof and a method for producing a BsAb antibody, thereby further utilizing the biology of mesenchymal stem cells.
  • the characteristic migrates to the treatment area, and the effect of immunotherapy is enhanced by the cell itself secreting BsAb antibody.
  • MSC Mesenchymal Stem Cell
  • the present invention constructs a "production factory" of a BsAb antibody and a targeted delivery system by genetically modifying the mesenchymal stem cells, and delivers the antibody to the lesion area by means of stem cells to complete stem cell mediated for diseases such as cancer. Stem-cell Driven Cancer-targeted Immunotherapy.
  • the present invention provides a genetically modified mesenchymal stem cell obtained by transfecting a mesenchymal stem cell with a non-viral vector carrying a BsAb antibody gene.
  • the genetically modified mesenchymal stem cells are capable of efficiently secreting bispecific single chain antibodies.
  • the mesenchymal stem cells may be derived from fat, umbilical cord, placenta, bone marrow, endometrium or pulp.
  • a BsAb antibody gene is carried by a non-viral gene carrier, and the non-viral vector may be selected from plasmid DNA (Plasmid DNA, hereinafter abbreviated as DNA) or microcircle plasmid DNA (Minicircle Plasmid). DNA, hereinafter abbreviated as MCDNA) and the like.
  • the BsAb antibody may be selected from the group consisting of antiCD19 ⁇ antiCD3, antiCD20 ⁇ antiCD3, antiEGFR ⁇ antiCD3, antiGPC3 ⁇ antiCD3, antiEpCAM ⁇ antiCD3, anticMet ⁇ antiCD3, antiEGFRvIII ⁇ antiCD3, antiIGF1R ⁇ antiCD3. , antiCD44v6 ⁇ antiCD3 or antiPDL-1 ⁇ antiCD3 and so on. Not limited to the sequence patterns described above, the same applies to the sequence pattern of the antiCD3 ⁇ anti "target".
  • the present invention provides a method for preparing the genetically modified mesenchymal stem cell, the method comprising:
  • the non-viral vector carrying the BsAb antibody gene is transfected into mesenchymal stem cells to complete genetic modification of the mesenchymal stem cells to obtain genetically modified mesenchymal stem cells.
  • the chemical transfection reagent may include: a lipofection reagent (such as Lipofectamine 2000 transfection reagent of Invitrogen), a polycation transfection reagent (such as polyethyleneimine, polyaminoester, chitosan, etc.). ), or inorganic nanoparticle transfection reagent, organic nanoparticle transfection reagent, or inorganic organic hybrid nanoparticle transfection reagent (such as magnetic nanoparticles, phosphate nanoparticles, etc.).
  • the electroporation transfection method can be a conventional electrorotation method.
  • the invention provides the use of the genetically modified mesenchymal stem cell for the production of a BsAb antibody.
  • the invention also provides a method of producing a BsAb antibody, the method comprising:
  • the genetically modified mesenchymal stem cells of the present invention are cultured to secrete a bispecific single chain antibody.
  • the culture conditions were 37 ° C, 5% CO 2 static culture.
  • the preferred medium is DMEM/F12 medium containing 10% fetal bovine serum.
  • the method for producing a BsAb antibody of the present invention further comprises the step of transfecting a non-viral vector carrying a BsAb antibody gene into mesenchymal stem cells to complete genetic modification of mesenchymal stem cells.
  • genetic modification to stem cells can be achieved by a chemical transfection method using a chemical transfection reagent or an electroporation transfection method.
  • the chemical transfection reagent may include: a lipofection reagent (such as Lipofectamine 2000 transfection reagent of Invitrogen), a polycation transfection reagent (such as polyethyleneimine, polyaminoester, chitosan, etc.), or Inorganic nanoparticle transfection reagent, organic nanoparticle transfection reagent, or inorganic organic hybrid nanoparticle transfection reagent (such as magnetic nanoparticles, phosphate nanoparticles, etc.).
  • the electroporation transfection method can be a conventional electrorotation method.
  • the method of producing a BsAb antibody of the present invention can be carried out according to any one of the following methods:
  • Method 1 Add 4 ⁇ g of plasmid DNA or microcircle MCDNA with antibody expression gene to an appropriate amount of opti-MEM medium, and let a final volume of 100 ⁇ l, let stand for 5 min; and add 10 ⁇ l of Lipofectamine 2000 gene transfection reagent (Invitrogen) to the appropriate amount.
  • the final volume was 100 ⁇ l, and after standing for 5 min, the plasmid solution and the transfection reagent solution were gently pipetted up and down by a pipette, and left at room temperature for 30 min to prepare a Lipofectamine 2000 gene carrier complex.
  • Mesenchymal stem cells were seeded in a 6-well plate at a density of 5 ⁇ 10 5 /ml per well (5 ⁇ 10 5 cells per well), and cultured in DMEM/F12 medium containing 10% fetal bovine serum. The cells were fused to 70%, 800 ⁇ l/well of opti-MEM medium was replaced, and then 200 ⁇ l of the gene vector complex was added to each well. After incubating for 4 hours at 37 ° C under 5% CO 2 , the culture was continued by changing the medium with DMEM/F12 medium containing 10% fetal bovine serum.
  • Method 2 4 ⁇ g of plasmid DNA or microcircle MCDNA with antibody expression gene is added to an appropriate amount of opti-MEM medium, the final volume is 100 ⁇ l, and allowed to stand for 5 min; and an appropriate amount of polycationic nanoparticles, such as ⁇ -into, is taken.
  • Ester-modified modified polyethyleneimine (patent application number 201310390436.1), dissolved in opti-MEM medium, the final volume of 100 ⁇ l is formulated into a transfection agent solution, after standing for 5min, the plasmid solution is transferred by pipette. The dye solution was gently mixed up and down, and allowed to stand at room temperature for 30 min to prepare a nanocomposite.
  • Human umbilical cord-derived mesenchymal stem cells were seeded in 6-well plates at a density of 5 ⁇ 10 5 /ml per well (5 ⁇ 10 5 cells per well), and cultured in DMEM/F12 containing 10% fetal bovine serum. Base culture, cells were fused to 70%, replaced with 800 ⁇ l/well of opti-MEM medium, and then 200 ⁇ l of nanocomposite was added to each well. After incubating for 4 hours at 37 ° C under 5% CO 2 , the culture was continued by changing the medium with DMEM/F12 medium containing 10% fetal bovine serum.
  • Method three The 18 ⁇ l Supplement solution [P3Primary Cell 4D-Nucleofector TM X Kit, Lonza Company] and 82 ⁇ l NucleofectorTM solution [P3Primary Cell 4D-Nucleofector TM X Kit, Lonza Company] and mix well to form an electrical-to-liquids (100 l System / electrostrictive rotor
  • the mesenchymal stem cells were digested with trypsin, detached from the culture dish wall, centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the mesenchymal stem cells were resuspended with 100 ⁇ l of electroporation to a density of 1 ⁇ 10 7 cells/ml; 6 ⁇ g of plasmid DNA or microcircle MCDNA with antibody-expressing gene was added to 100 ul of mesenchymal stem cell resuspension, and gently mixed with a pipette to mix the cells with the plasmid; The liquid was
  • stem cells are used as a BsAb antibody production plant, and BsAb antibodies can be continuously secreted for a long period of time.
  • the non-viral vector carrying the BsAb antibody gene is used, and the gene modification of the mesenchymal stem cells is completed by means of a chemical transfection reagent and an electrotransfection method, and the stem cells are constructed into a BsAb antibody.
  • “Production factory” and delivery system and secretion system, the present invention is compared with the clinical application technology of the existing BsAb, the main effects are as follows:
  • BsAb antibodies are mainly produced by genetic engineering. In order to prevent the harm of toxic substances such as immune heat sources, they must be purified by injection into the body, and BsAb antibodies are easily inactivated under temperature changes, thus increasing the difficulty of storage.
  • the present invention uses stem cells as a BsAb antibody production plant, and can inject the transfected stem cells to reduce the purification process;
  • BsAb antibodies can only rely on blood circulation to deliver BsAb antibodies to the target area.
  • existing studies have shown that the microenvironment of tumors and other lesions is very complicated, such as solid tumors often exhibit internal high pressure inside the tumor, which makes body fluids The antibody in it is difficult to enter the lesion area.
  • the stem cells are used as a BsAb antibody production plant, and the active chemotaxis and homing migration behavior of the tumors and the like by the mesenchymal stem cells are used to reach the target region to realize the delivery of the BsAb antibody;
  • BsAb antibody has a short half-life in vivo (less than 12 hours), thus reducing the probability of contact with target cells, so that the desired therapeutic effect cannot be formed; and the present invention uses stem cells as a BsAb antibody production plant, which can be used for a certain period of time. (Test test 72-96 hours) continuous expression, to a certain extent, the continuous supply of BsAb;
  • the present invention discards the conventional viral vector, and selects a non-viral gene vector to complete the stem cell genetic modification, thereby solving the problem of in vivo application feasibility;
  • plasmid DNA and microcircle MCDNA are used as gene carriers of BsAb to genetically modify stem cells, DNA plasmids and microcircle MCDNAs are stable in nature, and microcircle MCDNA is biosafety and can be stably expressed stably;
  • antibody secretion is achieved by means of stem cells, and stem cells as a "production plant” produce a bispecific single-chain antibody having a large molecular weight, which has a long-lasting biological effect, mobilizes a large number of immune cells to complete an immune attack on the target, and the stem cells can be returned.
  • the nest is located at the tumor or lesion site, thereby forming Stem-cell Driven Cancer-targeted Immunotherapy.
  • FIG. 1 is a schematic flow chart of a technical solution of the present invention.
  • Figure 2 is a map of the vector SZ63.pMC.ZY781.CMVmax.intron.bpA gene used in Example 1 of the present invention.
  • Figure 3 is a map showing the recombinant plasmid SZ66.pMC.ZY781.CMVmax.intron.CD20Bite.bpA after transformation in Example 1 of the present invention.
  • Figure 4 shows the results of western blot analysis of antiCD20 ⁇ antiCD3BsAb antibody in supernatant medium after 96 hours of transfection of human umbilical cord mesenchymal stem cells with Lipofectamine2000/plasmid DNA.
  • Figure 5 shows the western blot of antiCD20 ⁇ antiCD3BsAb antibody in the supernatant medium after 96 hours of transfection with umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells transfected with Lipofectamine2000-mediated plasmid DNA and microcircle MCDNA. Test results.
  • a non-viral vector carrying a BsAb antibody gene is transfected into a mesenchymal stem cell mainly by a chemical reagent-gene transfection method or an electroporation transfection method. Genetically modified mesenchymal stem cells are obtained, and then the genetically modified mesenchymal stem cells are cultured to secrete a bispecific single chain antibody.
  • Example 1 Chemical reagent transfection of human umbilical cord-derived mesenchymal stem cells
  • the antiCD20 ⁇ antiCD3 gene sequence (SEQ ID No. 1 is firstly constructed by molecular biological methods), and the sequence is transformed into a bispecific single-chain antibody sequence based on the humanized mAb anti-CD20 sequence.
  • the antiCD20 sequence is derived from: GA101 (Obinutuzumab, Roche Roche), and the antiCD3 part is derived from the patent US008076459) cloned into the vector SZ63.pMC.ZY781.CMVmax.intron.bpA. (Genetic and Cell of the Institute of Medicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences) Provided by the treatment laboratory, the detailed spectrum is shown in Figure 2, the sequence is shown in SEQ ID No.
  • the recombinant plasmid is the antiCD20 ⁇ antiCD3 sequence containing the antiCD20 ⁇ antiCD3 antibody expression gene plasmid DNA.
  • the specific preparation process is as follows:
  • PCR primer sequence (5'-3') forward: GAGCTAGCGCTACCGGTCGCCACCATGGGATGGAGCTGTATC (SEQ ID No. 3); reverse: TGAGTCGACCTAATGATGATGGTGATGA (SEQ ID No. 4) and containing the gene sequence of interest
  • the template (antiCD20 ⁇ antiCD3 sequence: SEQ ID No. 1) is amplified in a large amount by PCR (polymerase chain reaction), and then purified by TaKaRa gel recovery kit to obtain a higher purity target gene;
  • the vector SZ63.pMC.ZY781.CMVmax.intron.bpA and the target gene were treated by digestion, and the enzyme digestion was carried out at 37 ° C.
  • the enzyme cleavage sites were Nhe I and Sal I, and the TaKaRa gel recovery kit was used after digestion.
  • the carrier fragment and the target gene fragment with higher purity are obtained by gel recovery; the vector fragment and the target gene sequence after enzymatic digestion, the carrier fragment and the target gene fragment are mixed at a ratio of 1:3, and the TAKARA T4 is used at 16 ° C.
  • the ligase is ligated to the vector fragment and the gene of interest; the cloned recombinant plasmid DNA is obtained; the cloning recombinant plasmid DNA is ligated for transformation, and 10 ul of the recombinant plasmid is gently added to E.
  • coli competent cells gently mixed, and placed on ice for 30 minutes. Placed at 42 ° C for 90 seconds, then placed on ice for 5 minutes, added 500 ul LB medium to mix, placed at 37 ° C, 220 rpm for 1 hour, and finally coated with the card
  • the LB culture plate of natamycin is placed in a 37 ° C incubator for no more than 16 hours; then, the monoclonal antibody is picked up on the LB culture plate, and the plasmid is extracted and the plasmid is extracted according to the plasmid extraction kit method.
  • the plasmid DNA containing the expression gene of antiCD20 ⁇ antiCD3 antibody is obtained, that is, the recombinant plasmid SZ66.pMC.ZY781.CMVmax.intron.CD20Bite.bpA (6880 bp) shown in FIG. 3;
  • plasmid DNA with antiCD20 ⁇ antiCD3 antibody expression gene 4 ⁇ g was added to an appropriate amount of opti-MEM medium, the final volume was 100 ⁇ l, and allowed to stand for 5 min.
  • 10 ⁇ l of Lipofectamine 2000 gene transfection reagent (Invitrogen) was added to an appropriate amount of opti-MEM.
  • the final volume was 100 ⁇ l, and after standing for 5 min, the plasmid solution and the transfection reagent solution were gently pipetted up and down by a pipette, and left at room temperature for 30 min to prepare a Lipofectamine 2000/plasmid DNA complex.
  • Human umbilical cord-derived mesenchymal stem cells (supplied by Shenzhen Nanshan Hospital) were seeded in 6-well plates at a density of 5 ⁇ 10 5 /ml per well (5 ⁇ 10 5 cells per well) with 10% fetal bovine serum
  • the DMEM/F12 medium was routinely cultured, the cells were fused to 70%, the opti-MEM medium was changed to 800 ul/well, and then 200 ⁇ l of Lipofectamine 2000/plasmid DNA complex was added per well.
  • the medium was changed with DMEM/F12 medium containing 10% fetal bovine serum, and the culture supernatant was further cultured for 96 hours, and centrifuged at 10,000 rpm for 5 min at 4 ° C. The supernatant was stored at -80 ° C until use.
  • a negative control group was set as described above, and a PMAX plasmid containing EGFP (green fluorescent protein)-expressing gene (No. PMAX-eGFP, purchased from Lonza Co., Ltd.) was used to construct Lipofectamine 2000/DNA.
  • the complex was transfected, the stem cells were transfected, and the supernatant culture medium fraction of the cells collected after 96 hours was subjected to protein identification.
  • BsAb was transfected into human umbilical cord-derived mesenchymal stem cells, and the protein in the supernatant culture medium was subjected to western blot detection. The steps are as follows:
  • the cell supernatant sample was taken out from the -80 ° C refrigerator, and after melting at 4 ° C, 100 ⁇ l of the supernatant was taken, 25 ⁇ l of 5 ⁇ SDS-loading buffer was added, and the mixture was boiled at 100 ° C for 10 min. Cool to room temperature and load into the well. 80-120V electrophoresis for 90min.
  • the gel and membrane were fixed in a bio-Rad standard wet film transfer device with a transfer current of 300 mA and a film transfer time of 1 h. After the film was transferred, the film was taken out and washed in the TBST washing solution for 1 min, and added to 5% skim milk powder for 1 h. After the completion of the closure, wash 3 times with TBST, 5 min after each time, add Monoclonal The M2antibody (1:500, sigma-aldrich) primary antibody was incubated overnight at 4 °C.
  • the primary antibody was washed 3 times with TBST for 5 min each time, and a Goat anti-mouse-HRP (1:3000, Abcam) secondary antibody solution was added, and the mixture was incubated for 1 h at room temperature, and the primary antibody was washed 5 times with TBST for 5 min each time.
  • a Goat anti-mouse-HRP 1:3000, Abcam
  • Proteins were identified using an ANTI-FLAG antibody (purchased at Sigma-Aldrich).
  • the positive control group (Psitive control) was antiCD20 ⁇ antiCD3BsAb antibody standard; the negative control group (PMAX-eGFP) was used to carry the PMAX plasmid containing eGFP (green fluorescent protein) expression gene (purchased in Lonza) to construct Lipofectamine2000/
  • the DNA complex is targeted for stem cell transfection and protein identification of cell culture supernatants.
  • the transfection step was the same as that in Example 1.
  • the experimental group was constructed by constructing a plasmid DNA having an antiCD20 ⁇ antiCD3 expression gene into a Lipofectamine2000/DNA transfection complex for mesenchymal stem cell transfection, and identifying the protein of the cell culture supernatant.
  • the results showed that the antiCD20 ⁇ antiCD3BsAb antibody expressed by stem cells appeared in the same band position as the standard, and it was confirmed that the transfection was successful, and the stem cells could secrete antibodies.
  • Example 2 Electrotransfection of human umbilical cord-derived mesenchymal stem cells
  • plasmid DNA plasmid (see Example 1 for plasmid preparation) with antiCD20 ⁇ antiCD3 antibody expression gene was added to an appropriate amount of opti-MEM medium, the final volume was 100 ⁇ l, and allowed to stand for 5 min; and an appropriate amount of ⁇ -caprolactone was taken.
  • the modified modified polyethyleneimine (patent application number 201310390436.1) was dissolved in opti-MEM medium, and the final volume was 100 ⁇ l to prepare a transfection agent solution. After standing for 5 min, the plasmid solution and the transfection reagent were pipetted. The solution was gently mixed up and down, and allowed to stand at room temperature for 30 min to prepare a plasmid DNA nanocomposite.
  • Human umbilical cord-derived mesenchymal stem cells were seeded in 6-well plates at a density of 5 ⁇ 10 5 /ml per well (5 ⁇ 10 5 cells per well), using DMEM/F12 medium containing 10% fetal bovine serum. After the culture, the cells were fused to 70%, and the weiopti-MEM medium was replaced with 800 ul/well, and then 200 ⁇ l of the plasmid DNA nanocomposite was added to each well.
  • Example 4 Microcircular DNA (MCDNA) and plasmid DNA-mediated gene transfection for bone marrow mesenchymal stem cells
  • the MCDNA containing the antiCD20 ⁇ antiCD3 antibody-expressing gene used in the present Example was obtained by inducing a plasmid DNA carrying an antiCD20 ⁇ antiCD3 antibody-expressing gene to obtain an MCDNA carrying an antiCD20 ⁇ antiCD3 antibody-expressing gene.
  • the specific preparation process is as follows:
  • the plasmid DNA of the antiCD20 ⁇ antiCD3 antibody-expressing gene successfully constructed by the conventional method was added to 2 ml of TB medium (containing kanamycin 50 ⁇ g/ml) at 37 ° C.
  • the culture was shaken at 250 rpm, and after 1 hour, 1 ml of the bacterial solution was subjected to plasmid extraction according to the plasmid extraction kit method;
  • plasmid MCDNA with antiCD20 ⁇ antiCD3 antibody expression gene 4 ⁇ g was added to an appropriate amount of opti-MEM medium, the final volume was 100 ⁇ l, and allowed to stand for 5 min.
  • 10 ⁇ l of Lipofectamine 2000 gene transfection reagent (Invitrogen) was added to an appropriate amount of opti-MEM.
  • the final volume was 100 ⁇ l, and after standing for 5 min, the plasmid solution and the transfection reagent solution were gently pipetted up and down by a pipette, and left at room temperature for 30 min to prepare a Lipofectamine 2000/MC DNA transfection complex.
  • Human bone marrow mesenchymal stem cells (supplied by Shenzhen Nanshan Hospital) were seeded in 6-well plates at a density of 5 ⁇ 10 5 /ml per well (5 ⁇ 10 5 cells per well) with 10% fetal bovine serum. The cells were cultured in DMEM/F12 medium, and the cells were fused to 70%, and subjected to 800 ⁇ l/well of opti-MEM medium, and then 200 ⁇ l of a complex containing Lipofectamine 2000/MC DNA was added to each well.
  • the medium was changed with DMEM/F12 medium containing 10% fetal bovine serum, and the culture was continued for 96 hours (without changing the medium), and then the cell supernatant was collected. After centrifugation at 4 ° C, 10000 rpm for 5 min, the supernatant culture solution was collected for western blot detection. The detection results are shown in Figure 5.
  • Western blot results were used to identify proteins using the ANTI-FLAG antibody.
  • Western strip detection from left to right, 1 positive control group antiCD20 ⁇ antiCD3BsAb antibody standard; 2 using Lipofectamine2000/DNA transfection complex, transfecting umbilical cord mesenchymal stem cells with plasmid DNA with antiCD20 ⁇ antiCD3 antibody expression gene , expression of antiCD20 ⁇ antiCD3BsAb protein; 3 application of Lipofectamine2000/MCDNA transfection complex to transfected bone marrow mesenchymal stem cells with plasmid DNA of antiCD20 ⁇ antiCD3 antibody expression gene, antiCD20 ⁇ antiCD3BsAb protein expression; 4 application of Lipofectamine2000/DNA transfection Complex for transfection of bone marrow mesenchymal stem cells Plasmid DNA of antiCD20 ⁇ antiCD3 antibody-expressing gene, expression of antiCD20 ⁇ antiCD3BsAb protein in cell supernatant cultured at 96 hours; 5transfection of bone marrow mesenchymal

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Abstract

提供了一种基因修饰的间充质干细胞及其用于生产双特异性单链抗体(BsAb抗体)的方法，所述的基因修饰的间充质干细胞是利用携带双特异性单链抗体基因的非病毒载体转染间充质干细胞而得到的。还提供了通过基因修饰的间充质干细胞生产双特异性单链抗体的方法。

Description

基因修饰的间充质干细胞及其用于生产BsAb抗体的方法 技术领域
本发明是关于一种基因修饰的间充质干细胞及其用于生产BsAb抗体的方法，具体而言，本发明涉及基因修饰的间充质干细胞、其制备方法、以及通过基因修饰间充质干细胞生产双特异性单链抗体的方法。
背景技术
人体的免疫系统中，细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte，CTL)和杀伤性T细胞(Cytokine induced killer)扮演着重要的角色，它们拥有T细胞受体(T cell receptor，TCR)，能够对表达目标抗原的细胞进行特异识别与结合，并分泌穿孔素和颗粒酶，从而杀灭病原靶细胞。但是，癌细胞等病变区域常表现为抗原表达缺陷、缺少免疫粘附分子和共刺激分子等物质，使得机体免疫识别功能“丧失”。近年来，依照基因重组方法构建的双靶向抗体(Bispecificantibody,BsAb)，能够同时与T细胞和癌细胞特异性抗原结合，并激活T细胞免疫机制，实现“双靶向抗体介导T细胞重新定向的抗癌效应作用”(Anticancer effectors of BsAb-Retargeting T cell)。这种BsAb抗体协助功能性T细胞重新定位病变靶点，恢复了自体免疫功能。
目前临床治疗中，所使用的BsAb抗体主要通过体外基因工程生产，经蛋白纯化后由人体静脉注射，最终只能依靠血液循环将BsAb抗体递送到靶区域。这种方法获取的BsAb抗体极易在温度变化下失活、并且该治疗方法费用昂贵；同时，BsAb抗体半衰期较短，因此降低了与靶细胞的接触概率，以至于无法形成理想的治疗效果。
另一方面，应用干细胞产生细胞因子和蛋白已有相关报道。目前，对干细胞的基因修饰方法中大部分都选用病毒载体，如慢病毒、腺病毒、逆转录病毒。病毒载体转染虽然能提高干细胞基因修饰的效率，但由于病毒的安全性问题，使得这种方法存在一定的局限性，如对操作环境的要求严格，如感染后病毒基因可能随机整合到细胞基因组，从而破坏细胞基因组，造成致癌风险，况且病毒载体应用于动物体内其转染效率是不高的，因为机体本身会启动沉默病毒功能的机制。此外，现有的应用干细胞进行基因转染产生细胞因子和蛋白的研究方法所产生的蛋白和功能因子分子量相对较低，并且多见于应用核糖核酸类(RNA)如mRNA作为基因载体。
目前尚未见到应用干细胞生产分子量较大的双特异性单链抗体的类似报道。
发明内容
本发明的主要目的是应用间充质干细胞创建生物活性载体，提供一种基因修饰的间充质干细胞及其制备方法与用于生产BsAb抗体的方法，从而进一步可利用间充质干细胞的生物学特性迁移至治疗区域，通过细胞自身分泌BsAb抗体进而提高免疫治疗效果。
间充质干细胞(Mesenchymal Stem Cell，MSC)来源于间质组织，具备多向分化、免疫调节和自我复制更新功能。并且，在外界因素作用下，表现出趋向损伤和癌变病变组织的定向迁移能力(归巢性能)。本发明通过对间充质干细胞进行基因修饰，将干细胞构建成BsAb抗体的“生产工厂”和靶向性递送系统，借助干细胞实现将抗体递送至病变区域，完成针对如癌症等疾病的干细胞介导的细胞免疫治疗(Stem-cell Driven Cancer-targeted Immunotherapy)。
一方面，本发明提供了一种基因修饰的间充质干细胞，其是利用携带BsAb抗体基因的非病毒载体转染间充质干细胞而得到的。该基因修饰的间充质干细胞能够有效分泌双特异性单链抗体。
根据本发明的具体实施方案，本发明中，所述间充质干细胞可以来源于脂肪、脐带、胎盘、骨髓、子宫内膜或牙髓等。
根据本发明的具体实施方案，本发明中，是利用非病毒类基因载体承载BsAb抗体基因，这些非病毒载体可以选自质粒DNA(Plasmid DNA，下文缩写为DNA)或微环质粒DNA(Minicircle Plasmid DNA,下文缩写为MCDNA)等。
根据本发明的具体实施方案，本发明中，所述BsAb抗体可以选自antiCD19×antiCD3、antiCD20×antiCD3、antiEGFR×antiCD3、antiGPC3×antiCD3、antiEpCAM×antiCD3、anticMet×antiCD3、antiEGFRvIII×antiCD3、antiIGF1R×antiCD3、antiCD44v6×antiCD3或antiPDL-1×antiCD3等。不限于以上所述的序列模式，同样适用于antiCD3×anti“靶点”的序列模式。
另一方面，本发明还提供了所述的基因修饰的间充质干细胞的制备方法，该方法包括：
将携带BsAb抗体基因的非病毒载体转染至间充质干细胞，完成对间充质干细胞的基因修饰，得到基因修饰的间充质干细胞。
根据本发明的具体实施方案，本发明中，是利用化学转染试剂的化学转染方法、或电穿孔转染方法实现针对干细胞的基因修饰。具体而言，所述化学转染试剂可以包括：脂质体转染试剂(如Invitrogen公司的Lipofectamine2000转染试剂)，聚阳离子转染试剂(如聚乙烯亚胺、聚氨基酯、壳聚糖等)，或是无机纳米颗粒转染试剂、有机纳米颗粒转染试剂、或无机有机杂化纳米颗粒转染试剂(如磁性纳米颗粒、磷酸盐纳米颗粒等)。所述电穿孔转染方法可以为常规电转仪操作方法。
另一方面，本发明还提供了所述的基因修饰的间充质干细胞用于生产BsAb抗体中的应用。
另一方面，本发明还提供了一种生产BsAb抗体的方法，该方法包括：
培养本发明所述的基因修饰的间充质干细胞以分泌表达双特异性单链抗体。具体地，所述培养条件为37℃、5％CO2静置培养。其中优选的培养基为含10％胎牛血清的DMEM/F12培养基。采用本发明的方法，通常在培养72-96小时或更长的时间内可持续表达双特异性单链抗体，在一定程度上实现了BsAb的持续供给。
根据本发明的具体实施方案，本发明的生产BsAb抗体的方法还包括将携带BsAb抗体基因的非病毒载体转染至间充质干细胞完成对间充质干细胞的基因修饰的过程。如前所述，可以是利用化学转染试剂的化学转染方法、或电穿孔转染方法实现针对干细胞的基因修饰。所述化学转染试剂可以包括：脂质体转染试剂(如Invitrogen公司的Lipofectamine2000转染试剂)，聚阳离子转染试剂(如聚乙烯亚胺、聚氨基酯、壳聚糖等)，或是无机纳米颗粒转染试剂、有机纳米颗粒转染试剂、或无机有机杂化纳米颗粒转染试剂(如磁性纳米颗粒、磷酸盐纳米颗粒等)。所述电穿孔转染方法可以为常规电转仪操作方法。
根据本发明的更具体实施方案，本发明的生产BsAb抗体的方法可以按照下述方法中的任意一种进行：
方法一：将具有抗体表达基因的质粒DNA或微环MCDNA 4μg加入至适量的opti-MEM培养基中，终体积为100μl，静置5min；同时Lipofectamine2000基因转染试剂(Invitrogen公司)10μl加入至适量的opti-MEM培养基中，终体积为100μl，静置5min后，用移液器将质粒溶液和转染试剂溶液轻柔上下吹吸混合，室温放置30min，制成 Lipofectamine2000基因载体复合物。将间充质干细胞以每孔5×105个/ml的密度接种于6孔板中(每孔5×105个细胞)，用含10％胎牛血清的DMEM/F12培养基常规培养，细胞融合至70％，更换opti-MEM培养基800ul/孔，然后每孔加入200μl基因载体复合物。在37℃、5％CO2条件下孵育4小时后，用含10％胎牛血清的DMEM/F12培养基换液，继续培养。
方法二：将具有抗体表达基因的质粒DNA或微环MCDNA 4μg加入至适量的opti-MEM培养基中，终体积为100μl，静置5min；同时取适量的聚阳离子纳米颗粒,如ε-己内酯修饰的改性聚乙烯亚胺(专利申请号201310390436.1)，溶解于opti-MEM培养基中，终体积为100μl配制成转染剂溶液，静置5min后，用移液器将质粒溶液和转染试剂溶液轻柔上下吹吸混合，室温放置30min，制成纳米复合物。将人脐带来源的间充质干细胞以每孔5×105个/ml的密度接种于6孔板中(每孔5×105个细胞)，用含10％胎牛血清的DMEM/F12培养基培养，细胞融合至70％，更换为opti-MEM培养基800μl/孔，然后每孔加入200μl纳米复合物。在37℃、5％CO2条件下孵育4小时后，用含10％胎牛血清的DMEM/F12培养基换液，继续培养。
方法三：将18μl Supplement溶液[P3Primary Cell 4D-NucleofectorTM X Kit，Lonza公司]与82μl NucleofectorTM溶液[P3Primary Cell 4D-NucleofectorTM X Kit，Lonza公司]充分混匀，构成电转液(100μl体系/电转杯)；用胰酶消化间充质干细胞，使其从培养皿壁上脱落下来，离心(1000rpm，5min)，弃去上清，用100μl电转液将间充质干细胞重新悬浮，使其密度达到1×107个/ml；向100ul间充质干细胞重悬液中加入6μg具有抗体表达基因的质粒DNA或微环MCDNA，用移液枪轻轻混匀，使细胞同质粒充分混匀；将混合液加入到电转杯中，使用Lonza公司生产的AmaxaTM4D-NucleofectorTM细胞核转染仪，选取转染仪U-023程序进行电转；电转结束后，将提前预热的细胞培养液加入电转杯中，静置5min，用吸管轻轻吸出，加入到6孔盘培养皿中于37℃、5％CO2培养箱中静置培养，24小时后用含10％胎牛血清的DMEM/F12培养基更换，继续培养。
采用本发明的方法，应用干细胞作为BsAb抗体生产工厂，可在较长时期内持续分泌表达BsAb抗体。
综上所述，本发明中，主要是利用携带BsAb抗体基因的非病毒载体，借助化学转染试剂及电转染方法，完成对间充质干细胞的基因修饰，将干细胞构建成BsAb抗体的 “生产工厂”和递送系统与分泌系统，本发明与现有BsAb的临床应用技术相比，其有益效果主要表现为：
(1)现有临床应用中BsAb抗体主要通过基因工程生产，为防止免疫热源等有毒物质的危害必须经蛋白纯化后注入体内，并且BsAb抗体极易在温度变化下失活因此增加了保存难度。而本发明应用干细胞作为BsAb抗体生产工厂，可以将被转染干细胞注射，减少了纯化环节；
(2)临床应用中BsAb抗体只能依靠血液循环将BsAb抗体递送到靶区域，然而现有研究表明肿瘤等病灶的微环境十分复杂，如实体肿瘤常表现出瘤内的内部高压，这使得体液中的抗体难以进入病灶区域。而本发明将干细胞作为BsAb抗体生产工厂，利用间充质干细胞对肿瘤等病灶的主动趋化归巢迁移行为，到达目标区域，实现BsAb抗体的递送；
(3)BsAb抗体体内半衰期较短(小于12小时)，因此降低了与靶细胞的接触概率，以至于无法形成理想的治疗效果；而本发明应用干细胞作为BsAb抗体生产工厂，可以在一定时期内(试验检测72-96小时)持续表达，在一定程度上实现了BsAb的持续供给；
(4)本发明摒弃了常规的病毒载体，选用了非病毒基因载体来完成干细胞基因修饰，解决了体内应用可行性的问题；
(5)本发明中创新地使用了质粒DNA和微环MCDNA作为BsAb的基因载体对干细胞进行基因修饰，DNA质粒和微环MCDNA性质稳定，且微环MCDNA生物安全性高并且能持续稳定表达；
(6)本发明中借助干细胞实现抗体分泌，干细胞作为“生产工厂”产生分子量较大的双特异性单链抗体，生物效应持久，动员大量的免疫细胞完成对靶标的免疫攻击，并且干细胞能够归巢至肿瘤或病灶部位，从而可形成干细胞介导的细胞免疫治疗(Stem-cell Driven Cancer-targeted Immunotherapy)。
附图说明
图1为本发明的技术方案的流程示意图。
图2为本发明实施例1中所用载体SZ63.pMC.ZY781.CMVmax.intron.bpA基因图谱。
图3为本发明实施例1中改造后得到重组质粒SZ66.pMC.ZY781.CMVmax.intron.CD20Bite.bpA基因图谱。
图4为Lipofectamine2000/质粒DNA对人源脐带间充质干细胞转染96小时后，上清培养基中antiCD20×antiCD3BsAb抗体的western blot检测结果。
图5为针对脐带间充质干细胞、骨髓间充质干细胞，应用Lipofectamine2000介导质粒DNA和微环MCDNA进行基因转染，转染96小时后，对上清培养基中antiCD20×antiCD3BsAb抗体的western blot检测结果。
具体实施方式
为了更清楚地理解本发明的实质，下面通过具体实施例并配合附图进一步详细说明本发明，但本发明并不因此而受到任何限制。下列实施例中未注明具体条件的实验方法，通常按照所属领域的常规操作或按照制造厂商所建议的条件进行。
请参见图1所示，本发明的技术方案中，主要是利用化学试剂-基因转染方法、或是电穿孔转染方法，将携带BsAb抗体基因的非病毒载体转染至间充质干细胞，得到基因修饰的间充质干细胞，然后培养所述的基因修饰的间充质干细胞以分泌表达双特异性单链抗体。
实施例1、针对人脐带来源的间充质干细胞进行化学试剂转染
(1)制备Lipofectamine2000/DNA转染复合物
a、含有antiCD20×antiCD3抗体表达基因的质粒DNA的制备
本实施例中，先利用分子生物学方法将antiCD20×antiCD3基因序列(SEQ ID No.1，该序列是在人源化单抗anti-CD20序列的基础上改造而成双特异性单链抗体序列。antiCD20序列来源于：GA101(Obinutuzumab，罗氏Roche)，antiCD3部分来源于专利US008076459)克隆到载体SZ63.pMC.ZY781.CMVmax.intron.bpA.(由中国科学院深圳先进技术研究院医药所基因与细胞治疗研究室提供，其详细谱图如图2所示，序列如SEQ ID No.2(人工序列)所示，其中药物筛选标记为卡那霉素Kanamycin)，改造后得到重组质粒SZ66.pMC.ZY781.CMVmax.intron.CD20Bite.bpA(6880bp)如图3，该重组质粒即为含有antiCD20×antiCD3抗体表达基因质粒DNA antiCD20×antiCD3序列。具体制备过程如下：
利用上下游引物,其中PCR引物序列：(5’-3’)正向：GAGCTAGCGCTACCGGTCGCCACCATGGGATGGAGCTGTATC(SEQ ID No.3)；反向：TGAGTCGACCTAATGATGATGGTGATGA(SEQ ID No.4)和含有目的基因序列 模板(antiCD20×antiCD3序列：SEQ ID No.1)使用PCR(聚合酶链式反应)技术大量扩增该基因序列，而后利用TaKaRa胶回收试剂盒纯化回收得到较高纯度的目的基因；利用双酶切方法对载体SZ63.pMC.ZY781.CMVmax.intron.bpA及目的基因进行处理，在37℃条件下进行酶切，酶切位点为Nhe I和Sal I，酶切后利用TaKaRa胶回收试剂盒经过胶回收得到纯度较高的载体片段及目的基因片段；酶切后载体片段和目的基因序列，载体片段与目的基因片段按物质的量比1:3混匀，在16℃条件下借助TAKARA T4连接酶连接载体片段和目的基因；获得得到克隆重组质粒DNA；将连接得到克隆重组质粒DNA进行转化，10ul重组质粒轻轻加入到大肠杆菌感受态细胞，轻柔混匀，在冰上放置30分钟而后置于42℃条件下90秒钟，再置于冰上5分钟，加入500ul LB培养基混匀，置于37℃，220rpm条件下扩增1小时，最后涂在含有卡那霉素的LB培养板上，置于37℃培养箱中培养时间不超过16小时；而后，在LB培养板上挑取单克隆，按照质粒提取试剂盒方法，摇菌扩增并提取质粒，得到含有antiCD20×antiCD3抗体表达基因的质粒DNA，即图3所示重组质粒SZ66.pMC.ZY781.CMVmax.intron.CD20Bite.bpA(6880bp)；
b、制备Lipofectamine2000/DNA转染复合物的制备
将具有antiCD20×antiCD3抗体表达基因的质粒DNA4μg加入至适量的opti-MEM培养基中，终体积为100μl，静置5min；同时Lipofectamine2000基因转染试剂(Invitrogen公司)10μl加入至适量的opti-MEM培养基中，终体积为100μl，静置5min后，用移液器将质粒溶液和转染试剂溶液轻柔上下吹吸混合，室温放置30min，制成Lipofectamine2000/质粒DNA复合物。
(2)针对人脐带来源的间充质干细胞的化学试剂转染
人脐带来源的间充质干细胞(深圳南山医院提供)以每孔5×105个/ml的密度接种于6孔板中(每孔5×105个细胞)，用含10％胎牛血清的DMEM/F12培养基常规培养，细胞融合至70％，更换opti-MEM培养基800ul/孔，然后每孔加入200μl含有Lipofectamine2000/质粒DNA复合物。在37℃、5％CO2条件下孵育4小时后，用含10％胎牛血清的DMEM/F12培养基换液，继续培养96小时收集细胞上清培养液，4℃，10000rpm离心5min，取上清液保存于-80℃备用。
同时按照上述方法设定阴性对照组，应用含有eGFP(绿色荧光蛋白)表达基因的PMAX质粒(编号PMAX-eGFP，购置于Lonza公司)构建Lipofectamine2000/DNA 转染复合物，对干细胞进行转染，96小时后采集的细胞的上清培养液成分，进行蛋白鉴定。
(3)对针对人脐带来源的间充质干细胞转染BsAb，对细胞的上清培养液中蛋白进行western blot检测。其步骤如下：
(A)SDS-PAGE凝胶配制，先制备分离胶，待胶凝固后，配制浓缩胶，按照下述配方配制：
10％分离胶：
Figure PCTCN2015099177-appb-000001
浓缩胶：
Figure PCTCN2015099177-appb-000002
(B)制样及电泳
从-80℃冰箱取出细胞上清样品，4℃融化后，取100μl上清，加入25μl5×SDS-loading buffer，混匀后100℃煮10min。冷却至室温，上样至加样孔内。80-120V电泳90min。
(C)转膜后封闭孵育抗体
PVDF用甲醇活化5min后，将胶和膜固定在bio-Rad的标准湿法转膜装置内，转膜电流为300mA，转膜时间1h。转膜完毕，将膜取出在TBST洗涤液中洗涤1min，加 入至5％的脱脂奶粉中封闭1h。封闭完成后，用TBST洗涤3次，每次5min后，加入Monoclonal
Figure PCTCN2015099177-appb-000003
M2antibody(1:500，sigma-aldrich)一抗于4℃孵育过夜。次日，用TBST洗涤一抗3次，每次5min，加入Goat anti-mouse-HRP(1:3000，Abcam)二抗溶液，室温孵育1h，用TBST洗涤一抗5次，每次5min。
(D)ECL法检测蛋白
500μl Thermo Pierce ECL Western Blotting Substrate显影底物加至膜上，用凝胶成像仪捕捉化学发光条带，成像30s-2min。
检测结果如图4所示。应用ANTI-FLAG抗体(购置于Sigma-Aldrich公司)对蛋白进行识别。阳性对照组(Psitive control)为antiCD20×antiCD3BsAb抗体标准品；阴性对照组(PMAX-eGFP)，其为应用携带含有eGFP(绿色荧光蛋白)表达基因的PMAX质粒(购置于Lonza公司)，构建Lipofectamine2000/DNA复合物针对干细胞转染，细胞培养上清液的蛋白鉴定。其中转染步骤与实施例1相同；实验组为将具有antiCD20×antiCD3表达基因的质粒DNA构建成Lipofectamine2000/DNA转染复合物针对间充质干细胞转染，细胞培养上清液的蛋白鉴定。结果显示干细胞表达的antiCD20×antiCD3BsAb抗体与标准品出现在同一个条带位置，证实转染成功，干细胞可以分泌抗体。
实施例2、针对人脐带来源的间充质干细胞进行电转染
将18μl Supplement溶液[P3Primary Cell 4D-NucleofectorTM X Kit，Lonza公司]与82μl NucleofectorTM溶液[P3Primary Cell 4D-NucleofectorTM X Kit，Lonza公司]充分混匀，构成电转液(100μl体系/电转杯)；用胰酶消化间充质干细胞(深圳南山医院提供)，使其从培养皿壁上脱落下来，离心(1000rpm，5min)，弃去上清，用100μl电转液将间充质干细胞重新悬浮，使其密度达到1×107个/ml；向100μl间充质干细胞重悬液中加入6μg具有antiCD20×antiCD3-BsAb抗体表达基因的质粒DNA质粒(质粒制备参见实施例1)，用移液枪轻轻混匀，使细胞同质粒充分混匀；将混合液加入到电转杯中，使用Lonza公司生产的AmaxaTM4D-NucleofectorTM细胞核转染仪，选取转染仪U-023程序进行电转；电转结束后，将提前预热的细胞培养液加入电转杯中，静置5min，用吸管轻轻吸出，加入到6孔盘培养皿中于37℃、5％CO2培养箱中静置培养，24小时后用含10％胎牛血清的DMEM/F12培养基更换，而后加入10％胎牛血清的DMEM/F12培养基，在37℃、5％CO2条件下继续培养96小时(期间不进行换液)，而后收集细胞上清培养液， 4℃，10000rpm离心5min，收集上清培养液进行蛋白鉴定。检测结果参见图5。
实施例3、针对人脐带来源的间充质干细胞进行纳米复合物介导转染
将具有antiCD20×antiCD3抗体表达基因的质粒DNA质粒(质粒制备参见实施例1)4μg加入至适量的opti-MEM培养基中，终体积为100μl，静置5min；同时取适量的ε-己内酯修饰的改性聚乙烯亚胺(专利申请号201310390436.1)溶解于opti-MEM培养基中，终体积为100μl配制成转染剂溶液，静置5min后，用移液器将质粒溶液和转染试剂溶液轻柔上下吹吸混合，室温放置30min，制成质粒DNA纳米复合物。
人脐带来源的间充质干细胞以每孔5×105个/ml的密度接种于6孔板中(每孔5×105个细胞)，用含10％胎牛血清的DMEM/F12培养基培养，细胞融合至70％，更换weiopti-MEM培养基800ul/孔，然后每孔加入200μl含有质粒DNA纳米复合物。在37℃、5％CO2条件下孵育4小时后，用含10％胎牛血清的DMEM/F12培养基换液，而后在37℃、5％CO2、含10％胎牛血清的DMEM/F12培养基继续培养96小时(期间不进行换液)，而后收集细胞上清培养液，4℃，10000rpm离心5min，收集上清培养液进行蛋白鉴定。检测结果参见图5。
实施例4、针对骨髓间充质干细胞进行微环DNA(MCDNA)和质粒DNA介导的基因转染
(1)制备Lipofectamine2000/MCDNA转染复合物
a、含有antiCD20×antiCD3抗体表达基因的MCDNA的制备
本实施例中所用的含有antiCD20×antiCD3抗体表达基因的MCDNA，为针对含有antiCD20×antiCD3抗体表达基因的质粒DNA经过诱导得到携有antiCD20×antiCD3抗体表达基因的MCDNA。其具体制备过程如下：
将常规方法成功构建的antiCD20×antiCD3抗体表达基因的质粒DNA的大肠杆菌(菌株名称DH5α，全式金公司)种加入2ml的TB培养基(含有卡那霉素50μg/ml)，在37℃，250rpm条件下震荡培养，5小时后对其中1ml菌液按照质粒提取试剂盒方法进行质粒提取；
将上述质粒DNA的剩余大肠杆菌液100ul加入到400ml的TB培养基中(含有卡那霉素50μg/ml)，于37℃，250rpm条件下震荡培养过夜不超过16小时；
而后向400ml携带有上述质粒DNA的大肠杆菌液中加入微环诱导混合试剂(400ml  LB培养基中含有1N NaOH，20％Arabinose，具体参见表1)，再置于32℃，250rpm条件下振荡摇菌5-8小时；将菌液置于6000-rpm，4℃，离心10分钟，而后利用Qiagen质粒提取大提试剂盒进行质粒提取，最后获得含有antiCD20×antiCD3抗体表达基因的MCDNA。
表1、微环诱导混合试剂(ml)
Figure PCTCN2015099177-appb-000004
b、Lipofectamine2000/MCDNA转染复合物的制备
将具有antiCD20×antiCD3抗体表达基因的质粒MCDNA4μg加入至适量的opti-MEM培养基中，终体积为100μl，静置5min；同时Lipofectamine2000基因转染试剂(Invitrogen公司)10μl加入至适量的opti-MEM培养基中，终体积为100μl，静置5min后，用移液器将质粒溶液和转染试剂溶液轻柔上下吹吸混合，室温放置30min，制成Lipofectamine2000/MCDNA转染复合物。
(2)针对人源骨髓间充质干细胞的化学试剂转染
人源骨髓间充质干细胞(深圳南山医院提供)以每孔5×105个/ml的密度接种于6孔板中(每孔5×105个细胞)，用含10％胎牛血清的DMEM/F12培养基培养，细胞融合至70％，换opti-MEM培养基800ul/孔，然后，每孔加入200μl含有Lipofectamine2000/MCDNA的复合物。在37℃、5％CO2条件下孵育4小时后，用含10％胎牛血清的DMEM/F12培养基换液，继续培养96小时(期间不进行换液)，而后收集细胞上清培养液，4℃，10000rpm离心5min，收集上清培养液进行western blot检测实验，检测结果参见图5。
参见图5所示，western blot检测结果，应用ANTI-FLAG抗体对蛋白进行识别。Western条带检测，从左至右分别为①阳性对照组antiCD20×antiCD3BsAb抗体标准品；②应用Lipofectamine2000/DNA转染复合物，针对脐带间充质干细胞转染具有antiCD20×antiCD3抗体表达基因的质粒DNA，antiCD20×antiCD3BsAb蛋白表达结果；③应用Lipofectamine2000/MCDNA转染复合物针对骨髓间充质干细胞转染具有antiCD20×antiCD3抗体表达基因的质粒DNA，antiCD20×antiCD3BsAb蛋白表达结果；④应用Lipofectamine2000/DNA转染复合物针对骨髓间充质干细胞转染具有 antiCD20×antiCD3抗体表达基因的质粒DNA，96小时检测的细胞上清培养液中antiCD20×antiCD3BsAb蛋白表达结果；⑤应用纳米DNA转染复合物针对骨髓间充质干细胞转染具有antiCD20×antiCD3抗体表达基因的质粒DNA，96小时检测的细胞上清培养液中antiCD20×antiCD3BsAb蛋白表达结果。结果表明干细胞表达的antiCD20×antiCD3BsAb抗体与标准品出现在同一个条带位置，证DNA和MCDNA在转染试剂和纳米粒子体系下转染成功，干细胞可以分泌抗体。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (10)

  1. 一种基因修饰的间充质干细胞，其是利用携带BsAb抗体基因的非病毒载体转染间充质干细胞而得到的。
  2. 根据权利要求1所述的基因修饰的间充质干细胞，其中，所述间充质干细胞来源于滑膜、骨骼、肌肉、肺脏、肝脏、胰腺、脂肪、脐带、脐带血、胎盘、骨髓、子宫内膜或牙髓；以及包括以iPS技术诱导成体细胞转化为的间充质干细胞。
  3. 根据权利要求1所述的基因修饰的间充质干细胞，其中，所述携带BsAb抗体基因的非病毒载体选自：质粒DNA、微环质粒DNA或核糖核酸载体。
  4. 根据权利要求1所述的基因修饰的间充质干细胞，其中，所述BsAb抗体选自antiCD19×antiCD3、antiCD20×antiCD3、antiEGFR×antiCD3、antiGPC3×antiCD3、antiEpCAM×antiCD3、anticMet×antiCD3、antiEGFRvIII×antiCD3、antiIGF1R×antiCD3、antiCD44v6×antiCD3或antiPDL-1×antiCD3。
  5. 权利要求1～4任一项所述的基因修饰的间充质干细胞的制备方法，该方法包括：
    将携带BsAb抗体基因的非病毒载体转染至间充质干细胞，完成对间充质干细胞的基因修饰，得到基因修饰的间充质干细胞。
  6. 根据权利要求5所述的方法，其中，所述转染为利用化学转染试剂的化学转染方法、或电穿孔转染方法。
  7. 根据权利要求6所述的方法，其中，所述化学转染试剂包括：脂质体转染试剂，聚阳离子转染试剂，或是无机纳米颗粒转染试剂、有机纳米颗粒转染试剂、或无机有机杂化纳米颗粒转染试剂。
  8. 权利要求1～4任一项所述的基因修饰的间充质干细胞用于生产BsAb抗体中的应用。
  9. 一种生产BsAb抗体的方法，该方法包括：
    培养权利要求1～4任一项所述的基因修饰的间充质干细胞以分泌表达双特异性单链抗体。
  10. 根据权利要求9所述的方法，其中，所述培养条件为：37℃、5％CO2静置培养；优选的培养基为含10％胎牛血清的DMEM/F12培养基。
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