WO2011011973A1 - 抗cd20抗体fab片段与力达霉素的融合蛋白、制备方法及其用途 - Google Patents

抗cd20抗体fab片段与力达霉素的融合蛋白、制备方法及其用途 Download PDF

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WO2011011973A1
WO2011011973A1 PCT/CN2010/001141 CN2010001141W WO2011011973A1 WO 2011011973 A1 WO2011011973 A1 WO 2011011973A1 CN 2010001141 W CN2010001141 W CN 2010001141W WO 2011011973 A1 WO2011011973 A1 WO 2011011973A1
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fab
fusion protein
ldm
amino acid
ldp
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French (fr)
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杨纯正
甄永苏
熊冬生
邵荣光
朱祯
苗庆芳
程昕
张胜华
许元富
房虹
高瀛岱
金莲舫
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中国医学科学院医药生物技术研究所
中国医学科学院血液学研究所
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Priority to US13/387,531 priority Critical patent/US20120195895A1/en
Publication of WO2011011973A1 publication Critical patent/WO2011011973A1/zh

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775Apolipopeptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55Fab or Fab'
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Definitions

  • Anti-CD20 antibody Fab fragment fusion protein with lidamycin Anti-CD20 antibody Fab fragment fusion protein with lidamycin
  • the invention relates to the field of oncology and biopharmaceutics.
  • the present invention provides a fusion protein which can produce a tumor-targeting killing effect, a preparation method thereof and use thereof, and further provides an excellent drug candidate for targeted therapy of tumors.
  • Non-Hodgkin's lymphoma is a malignant tumor originating from lymphoid tissues, and its morbidity and mortality ranks fifth in malignant tumors.
  • conventional radiotherapy and chemotherapy have higher efficiency against NHL, they have poor selectivity. While killing tumor cells, they may damage certain types of normal cells in the body, and often have obvious toxic side effects. Therefore, tumor targeted therapy has become An important way to improve the treatment effect.
  • the target selection of targeted therapy is very important. It is known that most NHL originates from B lymphocytes, and more than 95% of B-cell NHL expresses CD20 antigen, while CD20 is only in pre-B lymphocytes. Expression in mature B lymphocytes, mature B lymphocytes, and activated B lymphocytes; no expression in plasma cells, lymphatic pluripotent stem cells, and other tissues, and CD20 antigens are exposed, and there is no free CD20 in human serum, so CD20 It can be used as an effective target for the treatment of B cell lymphoma.
  • anti-CD20 antibodies have recently been used to treat autoimmune diseases such as: rheumatoid Arthritis, systemic lupus erythematosus, etc. have become new research hotspots, which also suggests that the therapeutic application range of anti-CD20 antibodies will be further expanded.
  • autoimmune diseases such as: rheumatoid Arthritis, systemic lupus erythematosus, etc. have become new research hotspots, which also suggests that the therapeutic application range of anti-CD20 antibodies will be further expanded.
  • the number of cases treated with monoclonal antibodies increased Plus, the problem of drug resistance is becoming more and more obvious.
  • the I-labeled anti-CD20 murine antibody Bexxar and the 9 °Y-labeled Zevalin antibody developed in different ways, which are different from rituximab antibody, overcome drug resistance, but have high immunogenicity only once. Injection, and the toxicity and side effects are large, and the patient is poorly tolerated. Therefore, the development of small, highly effective anti-targeting drugs targeting CD20 has become a top priority. Brief description of the invention
  • the applicant envisages a strategy of combining miniaturized CD20 antibody Fab fragments (target vectors) with potent anti-tumor drugs (warheads).
  • the Fab fragment is composed of a heavy chain variable region VH, a CH1 in the constant region, a light chain variable region VL, and a constant region CL. It has a small molecular weight, strong penetrability, short half-life in vivo, and is easy to genetically engineer and pass. The advantages of mass production of bacterial fermentation. Moreover, due to the small molecular weight of Fab, low immunogenicity, it is not easy to produce HAMA reaction, and it can easily penetrate the dense tumor cell gap barrier and enter the deep part of solid tumor. Meanwhile, due to the lack of Fc fragment, Fc-mediated receptor binding is avoided.
  • LDM highly active "warhead” drug lindamycin
  • C-1027 or C1027 which was isolated from the soil of Qianjiang County, Hubei province, China.
  • the enediyne antibiotic produced by Streptomyces globi sporus (CMSCC No. 0704) is the macromolecular peptide antitumor antibiotic which has been reported to have the strongest killing effect on tumor cells.
  • LDM consists of two parts: one is an enedicyne structure chromophore (act ive enediyne, AE ), which has a cytotoxic effect but is unstable; the other is a prosthetic protein (LDP) consisting of 110 amino acid residues, which protects the chromophore from stability.
  • the chromophore and the prosthetic protein are bound by non-covalent bonds, and the combination of the two is specific and robust, and the pro-base protein and chromophore of LDM can be resolved and molecularly reconstructed.
  • LDM is suitable as a "warhead" drug with its unique molecular structure.
  • Applicants amplified the CHI fragment from the recombinant plasmid pCANTAB 5E Fcd20 Fab' containing the anti-CD20 antibody Fab fragment by genetic engineering method, and amplified the LDP gene from the shield pET30sngr ldp (Accession No. CGMCCNo. 2010), and then passed The Fab-LDP gene was obtained by S0E-PCR, and then the fragment was reassembled into the plasmid pCANTAB 5E Fcd20 Fab' excised with the CH1 gene to obtain the shield particle pCANTAB 5E-Fab-LDP containing Ant i-CD20 (Fab)-LDP.
  • the plasmid was transduced into the expression host strain, and the culture condition was optimized by changing the culture temperature, the medium composition and the culture time, and the soluble expression Fab-LDP fusion protein was obtained, and the fusion protein and the AE molecule were reassembled to obtain enhanced fusion.
  • Protein Fab - LDM Protein Fab - LDM.
  • the enhanced fusion protein Fab-LDM of the present invention retains the targeting of anti-CD20 antibody and the killing activity of LDM, and the Fab-LDM of the present invention exhibits a higher tumor than the same dose of Fab, LDM. Inhibitory effect.
  • a novel candidate targeted tumor therapeutic agent for tumor therapy which is a fusion of an anti-CD20 antibody Fab fragment and lidamycin, is provided.
  • Figure 1 shows the results of restriction endonuclease analysis of the recombinant expression plasmid pCANTAB 5E-Fab-LDP, wherein 1 is the DNA molecular weight standard; 2 is the recombinant plasmid pCANTAB 5E-Fab-LDP / apa l+sphl.
  • Figure 2a shows the results of SDS-PAGE analysis of the fusion protein Fab-LDP expression product, wherein
  • 1 is the recombinant strain pCANTAB 5E- Fab- LDP periplasmic cavity protein after non-reductive purification; 2 is a recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein non-reducing effluent;
  • FIG. 3 is the recombinant strain pCANTAB 5E- Fab-LDP periplasmic cavity protein non-reduction before loading; 4 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein i3L original purification; 5 is the recombinant strain pCANTAB 5E-Fab-LDP week The luminal protein reduces the effluent; 6 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein before the reduction is loaded.
  • Figure 2b is a result of Western blot analysis of the fusion protein Fab-LDP expression product, wherein
  • 1 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein non-reductive purification
  • 2 is the recombinant strain pCANTAB 5E-Fab-LDP periplasmic cavity protein non-reducing effluent
  • 3 is the recombinant strain pCANTAB 5E-Fab-LDP periplasm Cavity protein is not restored before loading.
  • Figure 3a shows the results of FACS analysis of the binding activity of anti-CD20 Fab and Fab-LDP to Ra j i cells, wherein ⁇ represents Fab-LDP; ⁇ represents anti-CD20Fab
  • Figure 3b shows the results of in vivo imaging of dynamic distribution of anti-CD20 Fab and Fab-LDP tumor-bearing mice.
  • Figure 4a shows the cytotoxic effect of the enhanced fusion protein Fab-LDM on Ra j i cells. among them,
  • Figure 4b shows the cytotoxic effect of the enhanced fusion protein Fab-LDM on Daud i cells. among them,
  • ADR Figure 4c shows the cytotoxic effect of the enhanced fusion protein Fab-LDM on K562 cells. Its Medium
  • ADR Figure 4d shows the comparison of IC50 of different fusion cells between the fusion protein Fab-LDM and LDM. among them,
  • Figure 5a shows mesh reinforcing ADR ⁇ fusion protein therapeutic effect of Fab-LDM trees early murine transplanted CD20-positive B cell lymphoma model, wherein
  • LDM 2nmol/kg.
  • Figure 5b shows the therapeutic effect of the enhanced fusion protein Fab-LDM on the late rat transplanted CD20+ B cell lymphoma model. among them,
  • stands for Fab- LDM 4nmol/kg
  • LDM 2nmol/kg.
  • Figure 6a shows the effect of the enhanced fusion protein Fab-LDM on body weight of rats. among them, -HH control
  • Figure 6c shows the effect of the enhanced fusion protein Fab-LDM on alanine aminotransferase (ALT) in mice. among them,
  • Figure 6d shows the effect of the enhanced fusion protein Fab-LDM on aspartate aminotransferase (AST) in rats. among them,
  • LDM low-density polystyrene-maleic anhydride copolymer
  • LDP-AE Low-density polystyrene-maleic anhydride
  • chromophore AE a chromophore AE is bound to a prosthetic protein.
  • the chromophore and the prosthetic protein are combined by non-covalent bonds, and the combination of the two is specific and robust.
  • the prosthetic proteins and chromophores of LDM can be resolved and molecularly reassembled.
  • AE as referred to herein means a chromophore having a chemical structure represented by the following formula I,
  • Fab as referred to herein is equivalent to "Anti-CD20 (Fab), "anti-CD20 Fab”, “Fab fragment of anti-CD20 antibody”, which is shown in positions 24-46 of SEQ ID NO: 1. Anti-CD20 antibody Fab fragment”.
  • Anti-CD20 (Fab)-LDP "a fusion protein of a Fab fragment of an anti-CD20 antibody and a lindamycin prosthetic protein", which is a fusion protein of SEQ ID NO: 1.
  • Fab - LDM as referred to herein is equivalent to "enhanced fusion protein Fab - LDM”"anti-CD20Fab-LDM” w Anti-CD20 (Fab) -LDM", "anti-CD20 antibody Fab and lidamycin”
  • a fusion protein which is a fusion protein of SEQ ID NO: 1, wherein a chromophore AE is bound to the lidomycinmycin prosthetic protein.
  • a fusion protein selected from one of the following sequences:
  • the vector of item 4 the vector being a plasmid.
  • a host strain comprising the vector of item 4.
  • the host strain of Project 6 which is deposited under the accession number CGMCC No. 3125, was sent to the China General Microorganisms Collection and Management Center on June 17, 2009, and named Escherichia coli named IHPAYZ.
  • a method of preparing a fusion protein of item 2 comprising the steps of:
  • step (c) purifying the fusion protein obtained in step (b),
  • step (e) it further comprises the step of determining the biological activity of the assembled fusion protein in step (d).
  • a pharmaceutical composition comprising a pharmaceutically effective amount of the fusion protein of item 1 or 2, optionally further comprising a pharmaceutically acceptable adjuvant.
  • lymphoma is a mouse lymphoma or a human B cell lymphoma.
  • a method of treating a disease comprising administering to a subject in need thereof an effective amount of a fusion protein of item 1 or 2.
  • the recombinant plasmid pCANTAB 5E Fcd20 Fab' contains the VH, VL and humanized CL, CH1 genes of anti-CD20 monoclonal antibody HI47, and the recombinant plasmid pCANTAB 5E Fcd20 Fab' contains only an apal restriction site in the CHI region, the applicant uses Infectious of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab') 2 fragment via induction. YinxingLiu, ZhenpingZhu et.al. Cancer Letters 205 (2004) 143-153) As a template, CH1 is obtained. The PCR primers were synthesized by Shanghai Yingjun Co., Ltd., and the corresponding restriction sites were introduced.
  • pCANTAB 5E Fcd20 Fab' was used as a template, P1 was used as a 5, a primer, and P2 was used as a 3, a primer, and PCR amplification was carried out under the following conditions: pre-denaturation at 94 ° C for 5 minutes, then 94. C was denatured for 1 minute, annealed at 56 ° C for 1 minute, and extended at 72 ° C for 1 minute for 25 cycles. After the last cycle, extend at 72 ° C for 10 minutes. A gene fragment A (about 324 bp) of a portion of CH1 starting with the apal cleavage site was obtained. Fragment A was subjected to 1.5% agarose gel electrophoresis and recovered by Bodatek A-type gel recovery kit.
  • Anti-CD20Fab' upstream primer PI Anti-CD20Fab' upstream primer PI
  • PCR amplification of LDP The recombinant plasmid pET30sngr ldp (Accession No. CGMCC No. 2010) containing the LDP gene was used as a template, P3 was used as a 5, primer, and P4 was used as a 3' primer, and PCR amplification was carried out. Reaction conditions: Pre-denaturation at 94 °C 5 Minutes, then 94. C denatured for 1 minute, 60. C annealed for 1 minute, 72. C stretched for 1 minute for 25 cycles. After the last cycle, extend at 72 ° C for 10 minutes. The gene fragment B of LDP (about 330 bp) was obtained. Fragment B was subjected to 1.5% agarose gel electrophoresis, and purified by Bodatek A-type gel recovery kit.
  • the purified fragment A (CHI) and fragment B (LDP) products were used for amplification.
  • the reaction conditions were: denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and extension at 72 ° C for 2 minutes for 10 cycles. A further extension of 72 ° C for 10 minutes yielded a small amount of CH1-1 inker-LDP template.
  • P1 and P4 primers were added and amplified.
  • the reaction conditions were: denaturation at 94 °C for 1 minute, annealing at 60 °C for 1 minute, 72. C extends for 2 minutes, a total of 30 cycles, and extends for another 10 minutes at 72 °C.
  • the Fab-LDP gene fragment C fragment A+B, about 669 bp
  • the reaction product C was subjected to 1% agarose gel electrophoresis, and recovered and purified by a Bodatek A-type gel recovery kit.
  • the recovered Fab-LDP fragment and the pCANTAB 5E Fcd20 Fab' vector were digested with apal and sphl, respectively, and then the reaction product was subjected to 1% agarose gel electrophoresis, and recovered and purified by Bodatek A-type gel recovery kit.
  • the obtained vector digested product and the digested product of the target gene were used in a ratio of 1:6 with Takara.
  • the company's T4 ligase 16 After 16 hours of C-ligation, the competent E.
  • the protein sequence of Fab-LDP is as shown in SEQ ID NO: 1, wherein position 1-23 is a signal peptide; 24-130 is a light chain variable region; 131-236 is a light chain constant region; 237-358 The position is the heavy chain variable region; the 359-466 position is the heavy chain CH1 region; the 467-471 position is G 4 S; and the 472-581 position is the lidamycin prosthetic protein.
  • G 4 S is a linker peptide consisting of 4 glycines and 1 serine.
  • Fab-LDP The DNA sequence of Fab-LDP is shown in SEQ ID NO: 2, wherein: 1-69 is the peptide sequence, 70-390 is the light chain variable region gene sequence; and 391-708 is the light chain constant region gene. Sequence; 709-1074 is the heavy chain variable region gene sequence; 1075-1398 is the heavy chain CH1 region gene sequence; 1399-1413 is G 4 S; 1414-1743 is the lidamycin prostaglandin gene sequence ; 1744-1746 is the stop codon.
  • SEQ ID NO: 2 The DNA sequence of Fab-LDP and Face Syndrome
  • a single colony of Escherichia coli HB2151 containing plasmid pC ANT AB 5 E Fab-LDP obtained in Example 1 was inoculated into 5 ml of 2 ⁇ ⁇ medium containing ampicillin (Amp) 100 g/ml, and shaken at a constant temperature. In a box of 37 * C, 200 rpm, shake overnight; transfer 500 ml of Amp 100 ⁇ g / ml of 2 ⁇ YT medium (1 liter of 2 ⁇ ⁇ medium containing 1. 6% trypsin, 1. 0% yeast pumping Extract, 0.5% sodium chloride, ⁇ 7.
  • the frozen cells were frozen and added with 50 ml of bacterial periplasmic protein extract (trihydroxyguanidinomethane 25 mmol / L, ethylenediaminetetraacetic acid 1 mmol / L, phenylmethylsulfonyl fluoride (PMSF) O.
  • bacterial periplasmic protein extract trihydroxyguanidinomethane 25 mmol / L, ethylenediaminetetraacetic acid 1 mmol / L, phenylmethylsulfonyl fluoride (PMSF) O.
  • Lmmol/L sucrose 20% (w/w, NaCl 200 mmol / L, pH 7.5), vortex and mix, placed in 4 lightly shake for 1 h. Centrifuge at 12000 rpm, 4 C for 20 minutes, take the supernatant.
  • the protein obtained in 2.1 was subjected to electrophoresis, and the electrophoresed gel was subjected to semi-dry electrorotation in a Bio-Rad electrorotation tank under the following conditions: a constant current of 0.7 mA/cm 2 and a time of 5 hours.
  • the polyvinylidene fluoride membrane (PVDF) after the end of electroporation was incubated with a primary antibody diluted 10 times in blocking solution, anti-LDP monoclonal antibody (Accession No. CGMCC No. 1849), labeled with horseradish peroxidase (HRP).
  • the goat anti-mouse IgG antibody was a secondary antibody and subjected to chromogenic analysis.
  • the in vitro immunofluorescence binding activity of Fab-LDP was determined by flow cytometry.
  • the 1106 th Raji cells were resuspended with different concentrations of FITC-labeled anti-CD20Fab fragment (Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab ') 2 fragment via induction of apoptosis.
  • FITC-labeled anti-CD20Fab fragment Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab ') 2 fragment via induction of apoptosis.
  • FITC labeling of Fab-LDP The Fab-LDP (5 mg/ml) to be cross-linked was dialyzed against the cross-linking reaction solution 4 X: 3 times to pH 9.0. Preparation method of cross-linking reaction solution: 7.56 g NaHC0 3 , 1.06 g Na 2 C0 3 , 7.36 g NaCl, and make up to 1 L with water. FITC was dissolved in DMSO at a concentration of 1 mg/ml. The FITC used for each cross-linking should be freshly prepared and stored in the dark.
  • the in vivo immunological binding activity of Fab-LDP was determined by a small animal in vivo imager. Take 5-6 weeks BALB/c 73i//2i rats, female, weight 18-20g (purchased from Beijing Weitong Lihua Experimental Animal Center, animal certificate number: SCXK (Beijing) 2007-0001). After systemic irradiation with Cs-ray (dose: 4 Gy/only), Raji or K562 cells were inoculated subcutaneously in the right axilla. The number of cells per inoculation was 2 X 1070.
  • Fab-LDP or anti-CD20 Fab can specifically bind to the tumor tissue at the CD20 antigen within 1 h; after 3 h, the tumor tissue center of the tumor-bearing mouse has begun to have Fab-LDP or anti-CD2 OFab enrichment, indicating The mAb has entered the center of the tumor; Fab-LDP or anti-CD20 Fab enrichment is still visible in the tumor tissue at 10 h, whereas LDP does not specifically bind to the tumor tissue site expressing the CD20 antigen (Fig. 3b).
  • Example 4 Preparation of Lidamycin and Determination of Relative Content of Active Chromophore AE 4. Preparation of Lida Zunsu (LDM)
  • the chromophore Compared with the LDM protein fraction, the chromophore has a smaller molecular weight and its theoretical content accounts for only 7.4% of lindamycin. Since AE is the active part of LDM, the prosthetic protein only protects the function of AE. Therefore, by measuring the relative content of AE in the total amount of chromophores, the activity of LDM products can be determined.
  • L Analysis of LDM by HPLC can measure the percentage of AE in the total amount of chromophores.
  • the specific method is:
  • the LDM preparation prepared as described above was dissolved in an HPLC mobile phase (acetonitrile: water: 23:77), and separated on a FPLC fast egg white iridometer by Waters semi-radial C4 semi-preparative column.
  • the eluent was acetonitrile: Water (23: 77), collected by an automatic collector, and the collected components were detected using an HPLC C4 analytical column.
  • the results of the analysis showed that the AE component of the LDM prepared by the inventors accounted for 90.63% of the total amount of LDM ⁇ L chromophore.
  • the LDM product was analyzed to meet the LDM quality control standard, and the AE high-content LDM product was lyophilized and stored in a refrigerator to enhance the preparation of the fusion protein Fab-LDM.
  • Example 5 Specific cytotoxicity of Fab-LDM on tumor cells
  • cytotoxic effect was determined by the thiazolyl blue (MTT) method. Take logarithmic growth phase of Ra ji or Daudi cells, count, 2 ⁇ 10 4 / wells in 96-well plates, at 37. After incubating for 12 hours in a C-containing C0 2 incubator, different concentrations of the drug Fab-LDM were added, and each drug concentration was set to 3 parallel wells. The culture was continued for 48 hours, centrifuged at 2000 rpm/min for 10 minutes, and the supernatant was discarded.
  • MTT thiazolyl blue
  • Cell viability (A dosing group - A blank group) / (A control group - A blank group) fusion protein was found to strengthen the Fab-LDM on Raj i, Daudi cells IC 5. Values are 0. 9x10- 1Q M and 0. 8x10-, IC 5 K562 cells no specific CD20 antigen. Is 3. 0 ⁇ 10 _10 ⁇ , Lidamycin IC 5 and the pair of Raj i, Daudi cells. The values are 3. 1x10 - 1D M and 2. 9x10 - , IC 5 for K562 cells without specific CD20 antigen. The value is 2. 8 ⁇ 10 " 10 ⁇ , indicating that Ant i-CD20 (Fab) -LDM retains the cytotoxicity of lidamycin and has selective killing effect on tumor cells. The specific results can be seen in Figure 4a. -d.
  • cytotoxicity was determined by the MTT assay. Take the patient's primary tumor cells, count, 2X10 4 /well in 96-well plates, at 37. C culture in an incubator containing 5% C0 2 After the hour, different concentrations of the drug Fab-LDM were added, and each drug concentration was set to 3 parallel wells. The culture was continued for 72 hours, centrifuged at 2000 rpm/min for 10 min, and the supernatant was discarded. Add 20 l of PBS dissolved MTT (5 mg/ml) per well, 37.
  • Fab-LDM retained the cytotoxicity of lidamycin and selectively inhibited the primary tumor cells of patients. It is worth mentioning that the primary cells of patients with resistance to rituximab are also very Good selective killing effect (Table 1).
  • Table 1 Growth inhibition of primary tumor cells in NHL patients by fusion protein Fab-LDM in vitro
  • NHL non-Linkin's lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • mice with a well-growth of 16-18 g were randomly divided into groups. After Cs irradiation, the Raj cells in the armpits of the rats were inoculated 2 ⁇ 7 7 /s within 24 h. After 7 days of feeding, the tail vein was administered intravenously, and 9 days later, the drug was administered again. The therapeutic effect of Fab-LDM on the subcutaneous lymphoma of the mouse was observed, and the growth curve of the mouse tumor was drawn. 2 ml ⁇ The control group was given intravenous saline 0.2 ml / only.
  • the other groups were given different doses of the fusion protein Fab-LDM, LDM and 4 nmol/kg of anti-CD20 Fab, all of which were injected into the tail vein, 0.2 ml/mouse.
  • the long diameter a and short diameter b of the tumor were measured every three days, and the animal body weight was recorded.
  • the tumor volume was calculated by the formula V-0. 5ab 2 and the tumor inhibition rate was calculated (Fig. 5a).
  • the other groups were given different doses of the enhanced fusion protein Fab-LDM and LDM, both of which were injected into the tail vein, 0.2 ml / each.
  • the long diameter a and the short diameter b of the tumor were measured every three days, and the animal body weight was recorded.
  • the results of the enhanced fusion protein Fab-LDM showed that Fab-LDM in 2mnol/kg and 4nmol/kg groups significantly inhibited or delayed the growth of transplanted lymphoma in mice, and showed a dose-free force. Dacmycin has a stronger tumor growth inhibition effect, showing a good therapeutic effect of Fab-LDM on advanced lymphoma.
  • mice weight (g) tumor volume inhibition rate
  • mice The changes in body weight and survival were observed, and adverse reactions or toxicity with significant physiological significance such as sputum dander, dehydration, lethargy, ataxia, and shortness of breath were recorded.
  • the body weight of the mice was weighed each time to assess the toxicity of the drug.
  • no adverse reactions with significant physiological significance such as sputum dander, dehydration, lethargy, ataxia, and shortness of breath were observed in the entire treatment window.
  • the PBS, Fab-LDM 2nmol/kg, Fab-LDM 4 leg 01/1 ⁇ and 0)1 1111101 /1 ⁇ groups of the mouse-resistant xenograft model showed no decrease in body weight and gradually increased.
  • the LDM 4nmol/kg group showed a decrease in body weight (Fig. 6a).
  • liver (kidney) lesion area statistics liver (kidney) lesion area / liver (kidney) total area. 7. 3 Detection of biochemical indicators such as liver function, kidney function and blood routine
  • the advantages and positive effects of the present invention are: using a combination of genetic recombination and molecular reconstitution, an enhanced fusion protein Fab-LDM of anti-CD20 Fab and anti-tumor antibiotic lindamycin is prepared, which not only retains the monoclonal antibody against CD20 antigen. It also has strong tumor cell-specific killing activity, and it also shows good anti-tumor effect in vivo. It has reached a new level in the miniaturization of tumor-targeted immunotherapy drugs and has a good application prospect.

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Description

抗 CD20抗体 Fab片段与力达霉素的融合蛋白、
制备方法及其用途 技术领域
本发明涉及肿瘤学和生物药学领域。 具体而言, 本发明提供 了可以产生靶向肿瘤杀伤作用的融合蛋白、其制备方法及其用途, 进而为肿瘤的靶向治疗提供了优良的候选药物。 技术背景
非霍奇金淋巴瘤 (NHL)是一种起源于淋巴组织的恶性肿瘤,其 发病率和死亡率已居恶性肿瘤第 5位。 常规的放疗和化疗虽然对 NHL有效率较高,但选择性差,在杀伤肿瘤细胞的同时也可能损害 体内某些类型的正常细胞, 常出现较明显的毒副反应, 因此肿瘤 靶向治疗已成为提高治疗效果的一条重要途径。
在肿瘤靶向治疗中, 靶向治疗的靶点选择非常重要, 已知大 多数 NHL起源于 B淋巴细胞, 95 %以上的 B细胞 NHL表达 CD20抗原, 而 CD20又仅在前 B淋巴细胞、 未成熟 B淋巴细胞、 成熟 B淋巴细胞、 激活 B淋巴细胞中表达; 在浆细胞、淋巴多能干细胞以及其它组织 均无表达, 且 CD20抗原比较暴露, 人体血清中也无游离的 CD20存 在, 因此 CD20可作为治疗 B细胞淋巴瘤的有效靶点。
在靶向治疗中使用最多的是与肿瘤相关抗原相关的单克隆抗 体。 自从 1997年美国 FDA批准抗 CD20人鼠嵌合抗体利妥昔抗体 (Ri tuximab)上市, 人们对抗体药物产生了极大的期望, 另外, 最 近利用抗 CD20抗体治疗自身免疫性疾病如: 类风湿性关节炎, 系统 性红斑狼疮等成为新的研究热点,这也提示抗 CD20抗体的治疗应用 范围将进一步得到扩展。 但随着使用单抗进行治疗的病例数的增 加, 药物抗性的问题也越来越明显。 随之发展起来的 I标记的抗 CD20鼠源性抗体 Bexxar和 9°Y标记的 Zeva l in抗体,作用机制与利妥 昔抗体不同, 克服了药物抗性, 但免疫原性大, 只能一次注射, 且毒副反应大, 患者耐受性差。 因此研制以 CD20为靶点的小型、 高效的抗 ^靶向药物成为当务之急。 发明简述
针对这种现状, 申请人设想釆用小型化 CD20抗体 Fab片段(靶 向载体) 与强效抗肿瘤药物 (弹头)相结合的策略。
在进行小型化抗体片段选择时,申请人选择了抗 CD20抗体 Fab 片段作为靶向载体。其中 Fab片段是由重链可变区 VH、恒定区中的 CH1和轻链可变区 VL、恒定区 CL组成,它具有分子量小、穿透力强、 体内半衰期短以及易于基因改造和可通过细菌发酵大量生产等优 点。 而且由于 Fab分子量小, 免疫原性低而不易产生 HAMA反应, 且 易穿透致密的肿瘤细胞间隙屏障, 可进入实体瘤深部; 同时因为 缺乏 Fc片段, 避免了 Fc介导的受体结合作用, 使得其快速向靶部 位集中; 此外还易于在体外进行基因改造, 通过基因重组技术, 在编码 Fab基因后面连接上活性蛋白基因, 并在受体中表达,产生 靶向融合蛋白。因此抗体 Fab片段作为肿瘤靶向药物的载体具有诱 人的前景。
在进行 "弹头" 选择时, 申请人选择了高活性的 "弹头" 药物 力达霉素 (LDM ) , 亦称 C- 1027或 C1027 , 是从中国湖北省潜江县土 壤中分离得到的一林由球孢链霉菌 (Streptomyces globi sporus , 菌种保藏编号: CGMCC No. 0704 )产生的烯二炔类抗生素, 是迄今报 道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素。 LDM 由两部分分子组成: 一为烯二炔结构的发色团 (act ive enediyne, AE ), 具有细胞毒作用, 但不稳定; 另一为 110个氨基酸残基组成的 辅基蛋白 (LDP ), 对发色团的稳定起保护作用。 发色团和辅基蛋白 通过非共价键结合, 两者结合具有特异性和牢固性, LDM的辅基蛋白 和发色团可以拆分和进行分子重建。 LDM以其独特的分子结构适合作 为 "弹头" 药物。
申请人通过基因工程方法 ,从含有抗 CD20抗体 Fab片段的重组质 粒 pCANTAB 5E Fcd20 Fab' 中扩增得到 CHI片段,从盾粒 pET30sngr ldp (保藏号 CGMCCNo. 2010 ) 中扩增得到 LDP基因, 然后通过 S0E-PCR 得到 Fab- LDP基因, 然后将该片段重新组装到切除了 CH1基因的质粒 pCANTAB 5E Fcd20 Fab' 中, 得到含有 Ant i-CD20 (Fab) - LDP的盾粒 pCANTAB 5E-Fab- LDP, 将该质粒转导到表达宿主菌株中, 通过改 变培养温度、 培养基成分和培养时间优化培养条件, 获得了可溶 性表达的 Fab - LDP融合蛋白, 将该融合蛋白与 AE分子重新组装, 得到强化融合蛋白 Fab - LDM。 在动物试验中, 本发明的强化融合 蛋白 Fab - LDM保留了抗 CD20抗体的靶向性和 LDM的杀伤活性,相较 相同剂量的 Fab、 LDM, 本发明的 Fab - LDM表现出更高的肿瘤抑制 效果。 由此提供了新型的、 由抗 CD20抗体 Fab片段和力达霉素融合 而成的可用于肿瘤治疗的候选靶向肿瘤治疗药物。 附图说明
图 1为重组表达质粒 pCANTAB 5E-Fab-LDP的限制性内切酶分 析结果, 其中, 1为 DNA分子量标准; 2为重组质粒 pCANTAB 5E-Fab-LDP /apa l+sphl。
图 2a为融合蛋白 Fab-LDP表达产物的 SDS-PAGE分析结果, 其 中,
1为重组菌株 pCANTAB 5E- Fab- LDP周质腔蛋白非还原純化后; 2为重组菌株 pCANTAB 5E-Fab-LDP 周质腔蛋白非还原流出 液;
3为重組菌株 pCANTAB 5E- Fab-LDP周质腔蛋白非还原上样前; 4为重组菌株 pCANTAB 5E-Fab-LDP 周质腔蛋白 i3L原纯化后; 5为重组菌株 pCANTAB 5E-Fab-LDP周质腔蛋白还原流出液; 6为重组菌株 pCANTAB 5E-Fab-LDP周质腔蛋白还原上样前。 图 2b为融合蛋白 Fab-LDP表达产物的 Wes tern印迹分析结果, 其中,
1为重组菌株 pCANTAB 5E-Fab-LDP周质腔蛋白非还原纯化后; 2为重组菌株 pCANTAB 5E-Fab-LDP周质腔蛋白非还原流出液; 3为重组菌株 pCANTAB 5E-Fab-LDP周质腔蛋白非还原上样 前。
图 3a为抗 CD20 Fab和 Fab-LDP与 Ra j i细胞的结合活性的 FACS 分析结果, 其中, ▼ 代表 Fab-LDP; □ 代表抗 CD20Fab
图 3b为抗 CD20 Fab和 Fab-LDP的荷瘤棵鼠体内动态分布的活 体成像仪观察结果。
图 4a表示强化融合蛋白 Fab-LDM对 Ra j i细胞的细胞毒作用。其 中,
→- Fab-LDM
→■ LDM
+ ADR β
图 4b表示强化融合蛋白 Fab- LDM对 Daud i细胞的细胞毒作用。 其中,
寺 LDM
- - Fab-LDM
ADR 图 4c表示强化融合蛋白 Fab-LDM对 K562细胞的细胞毒作用。其 中,
-ψ- Fab-LDM
-Β- LDM
ADR 图 4d表示强化融合蛋白 Fab-LDM与 LDM对不同细胞的 IC50的比 较。 其中,
□ Fab-LDM
^ LD
目 ADR β 图 5a表示强化融合蛋白 Fab-LDM对早期棵鼠移植性 CD20阳性 B细胞淋巴瘤模型的治疗作用, 其中,
*代表 PBS;
國代表抗 CD20Fab 4nmol/kg;
A f^Fab-LDM 2nmol/kg;
♦i^LDM 4nmol/kg;
▼i^Fab-LDM 4nmol/kg;
〇代表 LDM 2nmol/kg。
图 5b表示强化融合蛋白 Fab-LDM对晚期棵鼠移植性 CD20+B细 胞淋巴瘤模型的治疗作用。 其中,
•代表 PBS;
。代表抗 CD20Fab 4nmol/kg;
國 代表 Fab-LDM 2nmol/kg;
♦ 代表 LDM 4nmol/kg;
▲ 代表 Fab- LDM 4nmol/kg;
▼代表 LDM 2nmol/kg。
图 6a 表示强化融合蛋白 Fab-LDM对棵鼠体重的影响。 其中, -HH control
- - Fab 4nmol/kg
→- Fab-LDM 2nmol/kg
Fab-LDM 4nmol/kg
LDM 2nmol/kg
令 LDM 4nmol/kg 图 6b 表示强化融合蛋白 Fab-LDM对棵鼠白细胞的影响。 其中,
^SZ Fab 4nmol/kg
目 Fab-LDM 2nmol/kg
國 Fab-LDM 4nmol/kg 图 6c 表示强化融合蛋白 Fab-LDM对棵鼠丙氨酸转氨酶( ALT ) 的影响。 其中,
^ Fab 4nmol/kg
目 Fab-LDM 2nmol/kg
國 Fab-LDM 4nmol/kg 图 6d 表示强化融合蛋白 Fab-LDM对棵鼠天门冬氨酸转氨酶 ( AST ) 的影响。 其中,
E3 Fab 4nmol/kg
目 Fab-LDM 2nmol/kg
誦 Fab-LDM 4nmol/kg
发明详述
在本发明中所提及的术语均按下述定义来理解。
在本文中提到的 "LDM" 等同于 "LDP-AE" 、 "力达霉素" 、 "力达霉素辅基蛋白, 其中辅基蛋白上结合有发色团 AE" 。 发色 团和辅基蛋白通过非共价键结合,两者结合具有特异性和牢固性, LDM的辅基蛋白和发色团可以拆分和进行分子重新组装。
在本文中提到的 "AE"是指具有下式 I所示的化学结构的发色 团,
LDM^色团的化学名:
(2R, 7S, 9R, 10R) -7-氨基 -7,8- (2*-氯 -6* -羟基 -1*,4*-亚苯 基) -10- (4, -去氧 -4, -二曱氨基 -5,, 5, -二甲基-吡喃核糖基) -4, 8- 氧杂 -5-氧代 1, 11, 13-三烯 -15, 18-二炔 -三环 [7, 7, 3, 010'14] -2 -十 九碳醇 -2" ,3" -二氢 -7" -曱氧基 -2" -亚甲基- 3" -氧代 - ,4" -苯并恶咯 -5" -羧酸酯。 分子式: C43H42013N3C1
Figure imgf000009_0001
其由野生型力达霉素生产菌产生, 天然地结合于力达霉素的 辅基蛋白上, 而且可以通过在低温条件下用冷曱醇等有机溶剂处 理力达霉素的方式得到游离状态的发色团 AE, 该游离的发色团 AE 可以与去除了 AE的力达霉素辅基蛋白 LDP或者基因工程产生的 LDP (其上可以融合有其它蛋白片段或者不融合有其它蛋白片断)在 低温条件下组装成与天然力达霉素形式相同的活性形式, 这种重 新组装被称为 "强化" 。 在本文中提到的 "Fab" 等同于 "Anti-CD20(Fab)、 "抗 CD20Fab" 、 "抗 CD20抗体的 Fab片段" , 其是 SEQ ID NO : 1所示 的第 24 - 467位所示的抗 CD20抗体 Fab片段" 。
在 本 文 中 提 到 的 " Fab - LDP " 等 同 于 "antiCD20(Fab)-LDP" 、
"Anti-CD20 (Fab) -LDP" 、 "抗 CD20抗体的 Fab片段与力 达霉素辅基蛋白的融合蛋白",其是 SEQ ID NO: 1所示的融合蛋白。
在本文中提到的" Fab - LDM"等同于"强化融合蛋白 Fab - LDM" "anti-CD20Fab-LDM" wAnti-CD20 (Fab) -LDM" 、 "抗 CD20 抗体的 Fab与力达霉素的融合蛋白" , 其是 SEQ ID NO: 1所示的融 合蛋白, 其中力达霉素霉素辅基蛋白上结合有发色团 AE。
1. 融合蛋白, 其选自下述序列之一:
(a) 由 SEQ ID NO: 1所示的氨基酸序列构成的氨基酸序列;
(b)具有 SEQ IDN0: 1所示的氨基酸序列的生物学功能、 且 与 SEQ ID NO: 1所示的氨基酸序列具有 95%以上同源性的氨基酸 序列构成的氨基酸序列; 和
(c) 具有 SEQ IDN0: 1所示的氨基酸序列的生物学功能, 且 经过取代、 缺失或添加一个或几个氨基酸的 SEQ ID NO: 1所示的 氨基酸序列构成的氨基酸序列。
2. 项目 1的融合蛋白, 其还功能性地结合有式(I)所示结构 的发色团 AE:
Figure imgf000011_0001
力达霉素发色团结构式
( I)
3. 核酸分子, 其编码项目 1的融合蛋白的基因, 其选自下述 序列之一:
(a) SEQ ID NO: 2所示的编码 SEQ ID NO: 1的核苷酸序列;
(b) 编码项目 1中 (b) 的氨基酸序列的核苷酸序列;
(c) 编码项目 1中 (c) 的氨基酸序列的核苷酸序列; 和
(d) 因为密码子简并性而分别与 SEQ ID NO: 2不同、 但是 编码与 SEQ ID NO: 2所示序列相同的氨基酸序列的核苷酸序列。
4. 载体, 其可操作地连接有项目 3所述的核酸分子。
5. 项目 4的载体, 所述载体是质粒。
6. 宿主菌, 其包含项目 4所述的载体。
7. 项目 6的宿主菌, 其是保藏号为 CGMCC No.3125, 于 2009 年 06月 17日送交中国普通微生物菌种保藏管理中心保藏的名为 IHPAYZ的大肠埃希氏菌。
8. 制备项目 2的融合蛋白的方法, 包括以下步骤:
(a) 将抗 CD20抗体的 Fab基因与力达霉素辅基蛋白 LDP 基因可操作地连接到质粒 pCANTAB 5E 中, 得到重组表达质粒 pCANTAB 5E-Fab-LDP, ( b ) 在大肠杆菌 HB2151中诱导表达融合蛋白 Fab-LDP,
( c ) 纯化步骤(b ) 中获得的融合蛋白,
( d ) 使步骤(c ) 中获得的融合蛋白与式(I )的发色团组 装,
( e ) 任选地, 其还包括测定步骤 (d ) 中组装后的融合蛋 白的生物学活性的步骤。
9. 药物组合物, 其中含有药学有效量的项目 1或者 2所述的 融合蛋白, 任选地, 还含有药学上允许的佐剂。
10. 项目 1或考 2的融合蛋白在制备用于肿瘤靶向治疗的药物 中的用途。
11. 项目 10的用途, 所述药物用于靶向杀伤淋巴瘤细胞。
12. 项目 10的用途, 其中的淋巴瘤是棵鼠淋巴瘤或者人 B细 胞淋巴瘤。
13. 疾病治疗方法, 其包括向有此需要的受试者给予有效量 的项目 1或者 2的融合蛋白。
14. 项目 13的方法, 其中所述疾病是 CD20阳性的 B细胞淋巴
15. 项目 13的方法,其中所述疾病是非霍奇金淋巴瘤或弥漫 大 B细胞淋巴瘤。 下面通过实施例对本发明的具体实施方式进行阐述, 所述的 实施方式仅仅用来解释和说明本发明, 其并不限制本发明的保护 范围。 任何本领域技术人员根据公知的知识和现有技术的教导能 够想到的等价的变体都包含在本发明的保护范围中。 具体实施方式
实施例 1 重组表达质粒 pCANTAB 5E Fab-LDP的构建
PCR扩增 CH1:
由于重组质粒 pCANTAB 5E Fcd20 Fab' 含有抗 CD20单抗 HI47 的 VH、 VL及人源化 CL、CH1基因,且重组质粒 pCANTAB 5E Fcd20 Fab' 只有 CHI区含有一个 apal酶切位点, 所以申请人用含有 Fab, 基因 的重组质粒 pCANTAB 5E Fcd20 Fab' ( Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab' ) 2 fragment via induction of apoptosis. YinxingLiu, ZhenpingZhu et.al. Cancer Letters 205 (2004) 143-153 )作为模版, 获得 CH1。 PCR引物由上海英骏公司合成, 分别引入相应酶切位点。
具体地, 以 pCANTAB 5E Fcd20 Fab' 做为模板, 用 P1作为 5, 端引物, P2作为 3, 端引物, 进行 PCR扩增, 反应条件为: 94°C预 变性 5分钟, 然后 94。C变性 1分钟, 56°C 退火 1分钟, 72°C延伸 1分钟, 进行 25个循环。 最后一个循环后, 72°C延伸 10分钟。 得到 以 apal酶切位点开头的部分 CH1的基因片段 A (约 324bp) 。 将片 段 A进行 1.5%琼脂糖凝胶电泳, 并用博大泰克公司 A型胶回收试剂 盒回收纯化。
Anti-CD20Fab' 上游引物 PI:
5, -GCCTCC ACC AAGGGCCCATCGGTCTTCCCC- 3 ' ( SEQ ID No: 3 ) apal醉切位点
Anti-CD20Fab' 下游引物 P2:
5, -CGCGCTGCCACCGCCACCTGTGTGAGTTTTGTCACAAGA-3' (SEQ ID No: 4)
PCR扩增 LDP: 用含有 LDP 基因的重组质粒 pET30sngr ldp (保藏号 CGMCC No. 2010 )为模板, 用 P3作为 5, 端引物, 用 P4作为 3' 端引物, 进行 PCR扩增, 反应条件: 94°C预变性 5 分钟, 然后 94。C变性 1 分钟, 60。C 退火 1分钟, 72。C延伸 1分钟, 进行 25个循环。 最后 一个循环后, 72°C延伸 10分钟。得到 LDP的基因片段 B (约 330bp )。 将片段 B进行 1. 5%琼脂糖凝胶电泳, 并用博大泰克公司 A型胶回收 试剂盒回收纯化。
LDP上游引物 P3:
5, -ACAGGTGGCGGTGGCAGCGCGCCCGCCTTCTCCGTC3' ( SEQ ID No: 5 ) LDP下游引物 P4:
5, -GCGCGCATGCTCAGCCGAAGGTCAGAGCCAC-3' ( SEQ ID No: 6) s phi酶切位点
SOE-PCR扩增 Fab-LDP:
利用纯化的片段 A ( CHI )和片段 B ( LDP )产物, 进行扩增, 反 应条件: 94°C变性 1分钟, 60°C 退火 1分钟, 72°C延伸 2分钟, 共 10个循环,然后 72°C再延伸 10分钟,生成少量 CH1-1 inker-LDP 模板。 完成上步反应后, 补加 P1和 P4引物, 进行扩增, 反应条件: 94°C变性 1分钟, 60°C 退火 1分钟, 72。C延伸 2分钟, 共 30个循 环, 72°C再延伸 10分钟。 得到 Fab-LDP基因片段 C (片段 A+B,约 669bp ),
将反应产物 C进行 1 %琼脂糖凝胶电泳, 并用博大泰克公司 A 型胶回收试剂盒回收纯化。将回收得到的 Fab-LDP片段和 pCANTAB 5E Fcd20 Fab' 载体分别经 apal、 sphl酶切后, 将反应产物进行 1%琼 脂糖凝胶电泳, 并用博大泰克公司 A型胶回收试剂盒回收纯化。 将 得到的载体酶切产物与目的基因的酶切产物按 1 : 6的比例用 Takara 公司的 T4 连接酶 16。C连接 16 个小时后, 转化感受态大肠杆菌 HB2151 , 筛选出重组克隆质粒, 并进行菌液 PCR及酶切鉴定后, 测 序, 结果表明, 融合基因重组表达质粒的酶切结果和测序结果与预 期完全一致,序列正确, 命名为 pCANTAB 5E Fab-LDP, 菌体 PCR产 物的电泳图参见图 1。
其中 Fab - LDP的蛋白质序列如 SEQ ID N0: 1所示, 其中, 1-23 位为信号肽; 24-130位为轻链可变区; 131-236位为轻链恒定区; 237-358位为重链可变区; 359-466位为重链 CH1区; 467-471位为 G4S; 472-581位为力达霉素辅基蛋白。 其中 G4S为由 4个甘氨酸和 1 个丝氨酸组成的连接肽。
Fab-LDP 的 DNA序列如 SEQ ID NO: 2所示, 其中: 1-69位为 号肽基因序列, 70-390位为轻链可变区基因序列; 391-708位为轻 链恒定区基因序列; 709-1074位为重链可变区基因序列; 1075-1398 位为重链 CH1区基因序列; 1399- 1413位为 G4S; 1414-1743位为力 达霉素辅基蛋白基因序列; 1744-1746位为终止密码子。 实施例 2 Fab-LDP的表达与脸证
2. 1 Fab-LDP的表达
将实施例 1中获得的含有质粒 pC ANT AB 5 E Fab-LDP的大肠杆菌 HB2151的单菌落接种于 5 ml含氨苄青霉素 (Amp ) 100 g/ml的 2 χ ΥΤ培养基中, 在恒温摇瓶箱中 37 *C, 200rpm, 振荡培养过夜; 移入 500ml含 Amp 100 μ g/ml的 2 χ YT培养基( 1L 的 2 χ ΥΤ培养基含 1. 6% 胰化蛋白胨、 1. 0%酵母抽提物、 0. 5%氯化钠, ΡΗ 7. 4 ) 中, 37 Ό, 200rpm, 振荡培养 8h后, 在低温高速真空离心机上 6000rpm、 4 离 心 10分钟收集菌体, 将菌体重新悬浮于 1000 ml含 Amp 100 μ g/ml 及 ImM IPTG的 2 χ ΥΤ培养基, 30 , 200rpm,振荡培养 4 h; 8, 000rpm、 离心 10 分钟收集菌体, 冷冻于 -20 冰箱备用。
将冻存的菌体化冻, 加入 50ml细菌周质腔蛋白提取液(三羟曱 基氨基甲烷 25 mmol /L, 乙二胺四乙酸 1 mmol/L, 苯甲基磺酰氟 (PMSF) O. lmmol/L, 蔗糖 20% (w/w, NaCl 200 mmol /L, pH 7.5) , 振荡混匀, 置于 4 轻摇 1 h。 12000rpm, 4 C离心 20分钟, 取上清。 将提取物用 PBS透析 12 h后,在快速蛋白层析仪 ( FPLC )上进行纯化, 用结合緩沖液 (0.01 mol/L NaH2P04, 0.01 mol/L Na2HP04, 0.005% NaN3, pH 7.0)平衡亲和层析柱,上样, 再用结合緩沖液冲洗基线, 至基线 稳定后, 用洗脱緩冲液(0.1 mmol/L Glycine, pH 3,0)洗脱, 洗脱 液用中和緩冲液(1 mol/L Tris-HCl, 0.05% NaN3, pH 8.2)中和 pH 值。 然后用 12% SDS-PAGE分析外源蛋白表达情况, 结果表明, 经诱 导的重组菌株表达了大量外源蛋白,且 Fab-LDP的表达产物主要存在 于细菌可溶性周质腔中 (图 2a) 。
2. 2用 Western印迹法确认 Fab-LDP
将 2.1获得的蛋白进行电泳, 电泳后的凝胶在 Bio-Rad电转槽 中进行半干电转, 条件为: 恒电流 0.7mA/cm2, 时间为 5小时。 电转 结束后的聚偏二氟乙烯膜( PVDF )与用封闭液 10倍稀释的一抗即抗 LDP单抗(保藏编号 CGMCC No. 1849 )孵育,以辣根过氧化物酶( HRP ) 标记的羊抗小鼠 IgG抗体为二抗, 进行显色分析, 结果表明, 重组 菌株表达的确为 C-末端带有 LDP的重组融合蛋白 Fab-LDP(图 2b )。 将该含有质粒 pCANTAB 5E Fab-LDP的表达融合蛋白 Fab-LDP的大肠 埃希氏菌 (Escherichia coli )命名为 IHPAYZ, 于 2009年 06月 17日送交中国普通微生物菌种保藏管理中心保藏,保藏编号: CGMCC No.3125。 实施例 3 Fab-LDP的免疫学活性
通过流式细胞仪测定 Fab-LDP的体外免疫荧光结合活性。 将 1 106 个 Raji细胞重悬于含有不同浓度的 FITC标记的抗 CD20Fab片段 ( Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric (Fab' ) 2 fragment via induction of apoptosis. YinxingLiu , ZhenpingZhu et.al. Cancer Letters 205 (2004) 143-153 )或实施例 2获得的 Fab-LDP的 100 μ L PBS 溶液中, 放 置 1 h ,2000 g,离心 10分钟, 弃上清液, PBS 洗 3 次, FACS 测定 抗 CD2 OFab片段或 Fab-LDP结合 Ra j i细胞的阳性率。 证明相同浓度的 抗 CD20Fab片段及 Fab- LDP与 Raji细胞的结合活性基本相同, Fab-LDP 融合蛋白保留了与靶抗原特异性结合的能力。
Fab-LDP的 FITC标记: 将待交联的 Fab-LDP ( 5 mg/ml )对交联反 应液 4 X:下透析 3次,至 pH 9.0。交联反应液配制方法: 7.56 g NaHC03, 1.06 g Na2C03, 7.36 g NaCl, 加水定容至 1 L。 将 FITC溶于 DMSO中, 浓度为 lmg/ml。 每次交联使用的 FITC均应新鲜配制, 避光保存。 按 P: F (蛋白质: FITC) =1 mg: 150 的比例, 将 FITC緩慢加入于 Fab-LDP溶液中, 边加边轻轻晃动, 使其与 Fab-LDP混合均勾, 至平 面小摇床暗处 4 Ό轻轻摇动反应 12小时。 加入 5 mol/L的 NH4C1至终 浓度 50mmol/L, 4 "C终止反应 2 h。 将交联物在 PBS中透析 4次以上, 至透析液清亮。 交联物的鉴定: 蛋白浓度(mg/ml) -[A280 - 0.31 X A495]/1.4; F/P比例: 3.1 χ A495/ [A280 - 0.31 A495] , 该值应介 于 2.5- 6.5之间。
通过小动物活体成像仪测定 Fab-LDP的体内免疫结合活性。 取 5-6周 BALB/c 73i//2i棵鼠, 雌性, 体重 18-20g (购自北京维通利华实 验动物中心,动物合格证号: SCXK (京) 2007-0001 )。 经 Cs射线(剂 量: 4 Gy/只)全身照射后, 于右侧腋窝皮下接种 Raji或 K562细胞, 每只接种细胞数为 2 X 1070. 2ml β 肿瘤生长至 60-80 mm3时, 尾静脉 注射 Fab-LDP、 抗 CD20Fab、 LDP ( 16nmol/只) 。 分别于 1 h、 3 h、 及 10 h用活体动物成像系统观察 Fab-LDP在荷瘤鼠体内的动态分布。 结果发现 1 h 内 Fab-LDP或抗 CD20Fab即可特异性的结合在表达 CD20 抗原的肿瘤组织部位; 3 h后荷瘤棵鼠肿瘤组织中心已经开始有 Fab-LDP或抗 CD2 OFab富集,说明该单抗已经进入到肿瘤的中心部位; 10 h时肿瘤组织内仍可见 Fab-LDP或抗 CD20Fab富集, 而 LDP没有特异 性结合在表达 CD20抗原的肿瘤组织部位(图 3b ) 。 实施例 4 力达霉素的制备及其活性型发色团 AE的相对含量测定 4. 1力达尊素(LDM ) 的制备
将力达霉素产生菌 (CGMCC NO. 0135 )冷干管中加 0. 7 ml无盐 水, 使之形成菌悬液, 用白金耳接种于高氏 1号斜面培养基培养, 28 , 7- 10天, 表面生长白色气生菌丝, 取一小块接种于一级种子 100 ml/500 ml三角瓶培养 (发酵培养基成分为:淀粉 1%,玉米浆 0. 5%, 血胨 0. 5%, 葡萄糖 0. 5%, MgS04 0. 02% KI 0. 06%, 玉米面 1. 5%, CaC03 0. 4%, 自来水配制, pH 7. 0, 15磅消毒), 28 , 旋转摇床培养 48 h, 在转种 5%于 1000 ml/5000 ml立瓶中作为二级种子, 以相同发酵培养 基培养, 28Ό , 往返摇床培养 18 h, 上 200 L发酵罐, 装量为 100 L, 接种量 2%, 加 0. 03%泡敌为消沫剂, 罐压 0. 04, 28 °C , 搅拌 400转 / 分, 气流 1/1, pH 6. 5-7. 0, 发酵 96 h, 得到所需发酵液。 取发酵液 10 L, 离心取上清, 以 HC1调至 pH 4. 0, 加 ( NH4 ) 2S04 4. 5 Kg于 搅拌 3 h, 析出的力达尊素离心分离 (4Ό , 8000转 /分, 15 min ) , 所得的沉淀物加 200 ml冷水溶解, 透析, 再离心除去不溶物, 上清 液经羟基磷灰石柱吸附, 0. 001 M磷酸緩沖液( pH 6. 8 ) 洗脱, 活性 部分冷冻干燥, 得粗制品 1500 mg。 粗制品溶于水, 经 Sephadex G-75 柱层析, 活性部分冷冻干燥后, 得到 145 mg抗肿瘤高活性的力达霉 素白色粉末精制品。
4. 2活性型发色团 AE的相对含量测定
与 LDM蛋白部分相比,发色团分子量较小, 其理论含量仅占力达 霉素的 7. 4 %。 由于 AE是 LDM发挥作用的活性部分, 辅基蛋白仅有保 护 AE的功能, 因此一般通过测定 AE在发色团总量中的相对含量, 即 可以确定 LDM制品的活性高低。
釆用 HPLC 对 LDM进行分析可以测得 AE占发色团总量的百分比 值, 具体方法为:
将如上述制备的 LDM制品溶于 HPLC流动相(乙腈:水为 23 : 77 ), 在 FPLC快速蛋白色 i瞽仪上经 Wa ters径向加压 C4半制备柱分离,洗脱 液为乙腈: 水( 23: 77 ) , 自动收集器收集, 用 HPLC C4分析柱检测 收集到的各组分。
分析结果显示, 本发明人制备的 LDM, 其 AE组分占 LDM^L色团总 量的 90. 63%。通过分析确定该 LDM制品符合 LDM的质量控制标准, 为 AE高含量的 LDM精制品, 将该 LDM制品冻干, 置于- 冰箱保存, 以 便用于强化融合蛋白 Fab-LDM的制备。
4. 3强化融合蛋白 Fab-LDM的制备。
取高活性 LDM冻干品 10 mg, 加 5 ml 冷甲醇振摇 5分钟, -20°C放 置 1小时, 中间振摇 1次; 在 0。C, 12000转 /分钟离心 20分钟, 上清液 含发色团 AE, 沉降物为肽链, 重复提取 2次。 将含有发色团 AE的甲醇 液蒸发浓缩, - 70。C储存。 发色团 AE不稳定, 实验需低温(4°C ) 、 避光进行。
然后取实施例 2获得的 Fab- LDP融合蛋白分别溶于 PBS中, 加入 5 倍摩尔分子量的发色团-曱醇溶液(体积比为 50: 1 ) , 混合振摇, 室温放置 12小时。最后将混合液进行 PD-10柱层析,经 A280 nm和 A343 nm紫外监测后收集强化融合蛋白 Fab- LDM。 以下实施例所涉及的 Fab- LDM均按照本方法制备。 实施例 5 Fab-LDM对肿瘤细胞的特异性的细胞毒作用
5. 1 Fab-LDM对体外培养的肿瘤细胞的特异性的细胞毒作用 以噻唑蓝( MTT )法进行测定细胞毒作用。取对数生长期的 Ra j i 或 Daudi细胞、 计数, 2 χ 104个 /孔铺于 96孔板, 在 37。C含 5% C02 的培养箱中培养 12小时后加入不同浓度的药物 Fab-LDM, 每个药物 浓度设 3个平行孔。 继续培养 48小时, 2000rpm/分钟离心 10分钟, 弃上清。 每孔加入 20 μ ΐ PBS溶解的 MTT (5 mg/ml), 37°C继续培 养 4小时, 2000rpm/分钟离心 10分钟,轻轻弃去上清,加入 100 μ 1二甲基亚砜( DMS0 ),室温下摇床振摇 5分钟,酶标仪上测定 546 nm 光吸收值。 每次试验均设无药对照孔和无细胞对照孔各 3孔。 按下 述公式计算细胞的存活率及半数抑制浓度(IC5。)值:
细胞存活率 = ( A加药组 - A空白组) / ( A对照组 - A空白组) 结果发现强化融合蛋白 Fab-LDM对 Raj i、 Daudi细胞的 IC5。值 分别为 0. 9x10— 1Q M和 0. 8x10— , 对无特异性 CD20抗原的 K562细 胞的 IC5。值为 3. 0 χ10_10Μ, 而力达霉素对 Raj i、 Daudi细胞的 IC5。 值分别为 3. 1x10— 1D M和 2. 9x10— , 对无特异性 CD20抗原的 K562 细胞的 IC5。值为 2. 8 χ10"10Μ, 表明 Ant i-CD20 (Fab) -LDM较好的保留 了力达霉素的细胞毒作用,并对肿瘤细胞有选择性杀伤作用,具体结 果可以参见图 4a-d。
5. 2 Fab- LDM对病人原代肿瘤细胞的特异性的细胞毒作用 以噻唑蓝( MTT )法进行测定细胞毒作用。取病人原代肿瘤细胞, 计数, 2X104/孔铺于 96孔板, 在 37。C含 5% C02的培养箱中培养 12 小时后加入不同浓度的药物 Fab-LDM, 每个药物浓度设 3个平行孔。 继续培养 72小时, 2000rpm/min离心 l Omin, 弃上清。 每孔加入 20 l PBS溶解的 MTT (5 mg/ml) , 37。C继续培养 4小时, 2000rpm/min 离心 l Omin, 轻轻弃去上清, 加入 100 μ 1二曱基亚砜( DMS0 ), 室 温下摇床振摇 5分钟, 酶标仪上测定 546 nm光吸收值。 每次试验均 设无药对照孔和无细胞对照孔各 3孔。 按公式: 细胞存活率 = ( A 加药组 - A空白组) / ( A对照组 - A空白组) χ100%, 计算细胞的 存活率及半数抑制浓度 ( IC5。)值。
结果发现 Fab- LDM较好的保留了力达霉素的细胞毒作用, 并对 病人原代肿瘤细胞有选择性杀伤作用, 尤其值得一提的是, 对美罗 华耐药的病人原代细胞也有很好的选择性杀伤作用 (表 1 )。
表 1 : 融合蛋白 Fab-LDM体外对 NHL病人原代肿瘤细胞的生长抑制
Figure imgf000021_0001
缩写注释: NHL代表非霖奇金淋巴瘤; DLBCL代表弥漫大 B细胞淋巴瘤。 实施例 6 Fab-LDM对棵鼠移植性 CD20+B细胞淋巴瘤模型的治疗作 用
6. 1 Fab- LDM对早期棵鼠移植性 CD20+B细胞淋巴瘤 f莫型的治疗作 用
将生长状态良好的体重为 16- 18克的 5周龄 BALB/c棵鼠随机分 组, 经 Cs照射后, 24h内接种于棵鼠腋窝皮下 Raj i细胞 2 χ 107个 /只, 饲养 7天, 尾静脉注射给药, 9天后再次注射给药, 观察 Fab-LDM对棵 鼠皮下淋巴瘤的治疗作用, 绘制小鼠肿瘤的生长曲线。 对照组静脉 注射生理盐水 0. 2 ml/只。 其余各組分别给予不同剂量的强化融合蛋 白 Fab-LDM、 LDM及 4nmol/kg的抗 CD20Fab, 均为尾静脉注射, 0. 2 ml/ 只。 试验期间, 每三天测量一次肿瘤的长径 a和短径 b, 并记录动物 体重。 以公式 V-0. 5ab2计算瘤体积, 并计算抑瘤率(图 5a )。
强化融合蛋白 Fab- LDM的治疗结果表明, 2nmol/kg、 4nmol/kg 二 个剂量组的 Fab-LDM均能显著抑制或延迟棵鼠移植性淋巴瘤的生长, 均表现出比相应剂量游离力达霉素更强的肿瘤生长抑制作用, 提高 了力达霉素的治疗效果。 在实验第 39天的结果显示, 4nmol/kg和 2nmol/kg两个剂量组的 Fab-LDM的抑瘤率分别为 88%和 73 %, 强于相 应剂量 LDM组 49%和 69 %的抑瘤率。 在实验治疗期间, 动物体重有所 增加,——般状况良好, 表明动物可耐受所给剂量(表 2 )。 表 2. Fab- LDM对早期棵鼠移植性 CD20+B细胞淋巴瘤的生长抑制 作用
Figure imgf000022_0001
,与 LDM?f目比, AO. 05, 。与空白对照相比 *(0. 05, 。。与空白对照相比 AO. 01 ,
6. 2 Fab-LDM对晚期棵鼠移植性 CD20+B细胞淋巴瘤模型的治疗作 将生长状态良好的体重为 16-18克的 5周 BALB/c棵鼠随机分组, 经 Cs照射后, 24h内接种于棵鼠腋窝皮下 Raj i细胞 2 χ 107个 /只, 25 天后尾静脉注射给药, 9天后再次注射给药, 观察 Fab-LDM对棵鼠皮 下淋巴瘤的治疗作用, 绘制小鼠肿瘤的生长曲线(图 5b)。 对照组静 脉注射生理盐水 0. 2 ml/只。 其余各组分别给予不同剂量的强化融合 蛋白 Fab-LDM及 LDM, 均为尾静脉注射, 0. 2 ml /只。 试猃期间, 每三 天测量一次肿瘤的长径 a和短径 b,并记录动物体重。以公式 V=0. 5ab2 计算瘤体积,并计算抑瘤率。强化融合蛋白 Fab-LDM的治疗结果表明, 2mnol /kg、 4nmol /kg 二个剂量组的 Fab- LDM均能显著抑制或延迟棵 鼠移植性淋巴瘤的生长, 并且均表现出比相应剂量游离力达霉素更 强的肿瘤生长抑制作用,显示出 Fab-LDM对晚期淋巴瘤良好的治疗效 果。 在实验第 47天的结果显示,4nmol/kg和 2nmol/kg两个剂量组的 Fab-LDM的抑瘤率分别为 79. 2%和 67. 5 %, 强于相应剂量 LDM组 52. 1% 和 65 %的抑瘤率(表 3 )。 在实验治疗期间, 动物体重有所增加, 一 般状况良好, 表明动物可耐受所给剂量。
表 3. Fab-LDM对晚期棵鼠移植性 CD20+B细胞淋巴瘤的生长抑制作 用
组别 刑量 小鼠数量 体重 (g) 肿瘤体积 抑制率
(nmol/kg) (mm3) X (%)
开始 /结束 开始 /结束 .
对照 5/5 16. 5/20. 4 3255. 76
2 5/5 16. 8/20. 6 1559. 52 52. 1口
LDM
4 5/5 16. 5/20. 4 1136. 86 65πα
2 5/5 16. 2/20. 5 67. 5D*
Fab-LDM
4 5/5 17. 3/20. 6 79. 2°*
*与 LD咖匕, KQ. 05 , 。与空白对照相比 AO. 05 , 。。与空白对照相比 AO. 01. 实施例 7 Fab-LDM的毒性评估
7. 1体重和生存情况
观察小鼠体重变化和生存情况, 同时记录具有明显生理意义的 不良反应或毒性, 如糠枇性皮屑、 脱水、 嗜睡、 共济失调和呼吸急 促等。 每次测量时称小鼠体重, 以评估药物的毒性反应。 结果发现, 在整个治疗窗中, 各实验组均未观察到具有明显生理意义的不良反 应, 如糠枇性皮屑、 脱水、 嗜睡、 共济失调和呼吸急促等。 接受治 疗后棵鼠耐药移植瘤模型的 PBS 、 Fab-LDM 2nmol/kg 、 Fab-LDM 4腿01/1^及0)1 1111101 /1^组, 小鼠体重均无下降, 呈逐渐增长趋势, LDM 4nmol/kg组小鼠体重出现下降 (图 6a ) 。
7. 2损伤部位的病理学统计
实验结束后, 处死老鼠, 取心、 肝、 脾、 肺、 肾、 小肠等器官, 福尔马林浸泡过夜, HE染色。 采用 LEICA QWINV3 系统对各处理组的 多个脏器进行了 10张切片 50个视野的统计学分析, 计算各处理组各 器官的损伤面积。 结果发现 PBS 、 Fab-LDM 2nmol/kg 、 Fab-LDM 4nmol/kg及 LDM2 nmol/kg基本没有明显可见的病例损伤, 而 LDM 4nmol/kg组能引起轻微的肾小管肿胀, 肝组织炎细胞浸润, 脾脏造 血细胞减少等损伤 (表 4 ) 。 表 4: 损伤部位的病理学统计
Figure imgf000024_0001
*:脾脏损伤统计 = 4nmol /kg LDM处理组有核细胞数 /PBS 处理組有核细胞数。 **:肝脏(腎脏)损伤面积统计 =肝脏(肾脏)损伤面积 /肝脏(肾脏)总面积。 7. 3 肝功、 肾功、 血常规等生化指标的检测
实验结束后, 处死老鼠, 断头取血, 部分全血抗凝处理后检测 血常规的变化; 另一部分全血促凝处理后取血清检测肝功、 肾功的 变化。 结果发现, Fab-LDM 2nmol/kg、 Fab-LDM 4nmol/kg组对肝功、 肾功基本没有影响, 其中 Fab-LDM 4nmol/kg组引起白细胞轻微下降 (图 6b-d ) 。 发明效果:
本发明的优点与积极效果在于:应用基因重组和分子重建相结合 的方法, 制备了抗 CD20 Fab与抗肿瘤抗生素力达霉素的强化融合蛋 白 Fab- LDM,不仅保留了单抗对 CD20抗原的结合性, 还具有强烈的肿 瘤细胞特异性的杀伤活性, 在体内实验中也显示了良好的抗肿瘤疗 效。 在肿瘤靶向免疫治疗药物的小型化方面达到一个新水平, 具有 良好的应用前景。

Claims

权 利 要 求
1. 融合蛋白, 其选自下述序列之一:
(a) 由 SEQ ID NO: 1所示的氨基酸序列构成的氨基酸序列;
(b) 具有3£(310^: 1所示的氨基酸序列的生物学功能、 且 与 SEQ ID NO: 1所示的氨基酸序列具有 95%以上同源性的氨基酸 序列构成的氨基酸序列; 和
(c)具有 SEQ IDN0: 1所示的氨基酸序列的生物学功能, 且 经过取代、 缺失或添加一个或几个氨基酸的 SEQ ID NO: 1所示的 氨基酸序列构成的氨基酸序列。
2. 权利要求 1的融合蛋白, 其还功能性地结合有式( I )所示 结构的发色团 AE:
Figure imgf000026_0001
3. 核酸分子, 其编码权利要求 1 的融合蛋白的基因, 其选自 下述序列之一:
(a) SEQ ID NO: 2所示的编码 SEQ ID NO: 1的核苷酸序列; (b) 编码权利要求 1中 (b) 的氨基酸序列的核苷酸序列;
(c) 编码权利要求 1中 (c) 的氨基酸序列的核苷酸序列; 和
(d) 因为密码子简并性而与 SEQ ID NO: 2不同、 但是编码 与 SEQ ID NO: 2 所示序列相同的氨基酸序列的核苷酸序列。
4. 载体, 其可操作地连接有权利要求 3所述的核酸分子。
5. 权利要求 4的载体, 所述载体是盾粒。
6. 宿主菌, 其包含权利要求 4所述的载体。
7. 权利要求 6的宿主菌,其是保藏号为 CGMCCNo.3125,于 2009 年 06 月 17 日送交中国普通微生物菌种保藏管理中心保藏的名为 IHPAYZ的大肠埃希氏菌。
8. 制备权利要求 2的融合蛋白的方法, 包括以下步骤:
(a) 将抗 CD20抗体的 Fab基因与力达霉素辅基蛋白 LDP 基因可操作地连接到质粒 pCANTAB 5E 中, 得到重组表达质粒 pCANTAB 5E-Fab-LDP,
( b ) 在大肠杆菌 HB2151中诱导表达融合蛋白 Fab-LDP,
(c) 纯化步骤(b) 中获得的融合蛋白,
(d) 使步骤(c) 中获得的融合蛋白与式(I)的发色团组 装,
(e) 任选地, 其还包括测定步骤(d) 中组装后的融合蛋 白的生物学活性的步骤。
9. 药物组合物, 其中含有药学有效量的权利要求 1或者 2所 述的融合蛋白, 任选地, 还含有药学上允许的佐剂。
10. 权利要求 1或者 2的融合蛋白在制备用于肿瘤靶向治疗的 药物中的用途。
11. 权利要求 10的用途, 所述药物用于靶向杀伤淋巴瘤细胞。 权利要求 10的用途, 其中的淋巴瘤是棵鼠淋巴瘤或者人 B
13. 疾病治疗方法, 其包括向有此需要的受试者给予有效量的 权利要求 1或者 2的融合蛋白。
14. 权利要求 13的方法, 其中所述疾病是 CD20阳性的 B细胞 淋巴瘤。
15. 权利要求 13的方法, 其中所述疾病是非霍奇金淋巴瘤或弥 漫大 B细胞淋巴瘤。
PCT/CN2010/001141 2009-07-28 2010-07-27 抗cd20抗体fab片段与力达霉素的融合蛋白、制备方法及其用途 WO2011011973A1 (zh)

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