WO2017008736A1 - 抗体依赖性细胞介导的细胞毒性增强的奥法木抗体 - Google Patents

抗体依赖性细胞介导的细胞毒性增强的奥法木抗体 Download PDF

Info

Publication number
WO2017008736A1
WO2017008736A1 PCT/CN2016/089820 CN2016089820W WO2017008736A1 WO 2017008736 A1 WO2017008736 A1 WO 2017008736A1 CN 2016089820 W CN2016089820 W CN 2016089820W WO 2017008736 A1 WO2017008736 A1 WO 2017008736A1
Authority
WO
WIPO (PCT)
Prior art keywords
fucose
modified antibody
sugar chain
antibody
lymphoma
Prior art date
Application number
PCT/CN2016/089820
Other languages
English (en)
French (fr)
Inventor
于鹏展
吴伟
范林萍
Original Assignee
海思科医药集团股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 海思科医药集团股份有限公司 filed Critical 海思科医药集团股份有限公司
Priority to CN201680028044.2A priority Critical patent/CN108473570A/zh
Publication of WO2017008736A1 publication Critical patent/WO2017008736A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of genetic engineering and relates to a modified antibody which obtains a significant increase in antibody-dependent cell-mediated cytotoxicity (ADCC) by altering the sugar chain composition of the marketed Ofatumumab (Arzerra) mAb.
  • the invention also encompasses methods for the preparation of such modified antibodies and their use in the treatment of tumors, particularly chronic lymphomas with high expression of CD20, non-Hodgkin's lymphoma and diffuse large B-cell lymphoma.
  • CD20 also known as human B lymphocyte-restricted antigen
  • B lymphocytes matured from pre-B cells a hydrophobic transmembrane protein on B lymphocytes matured from pre-B cells.
  • One of the objects of the present invention is to enhance the biological activity, particularly antibody-dependent cell-mediated cytotoxicity (ADCC) activity, by altering the sugar chain composition of ofatumumab monoclonal antibody by regulating the metabolic pathway of cells, obtaining a A new antibody (modified antibody) with higher biological activity. Its anti-tumor activity has been improved in the present invention. In vitro tests at the cellular level and in vivo efficacy tests at the animal level were confirmed.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the CHO expression system replaces the NS0 expression system, thereby reducing or eliminating the Di- ⁇ 1,3GalGal sugar-modified component contained in the NS0 system when expressing the olfaximab, so that the monoclonal antibody of the present invention has lower immunogenicity.
  • Another object of the present invention is to provide a process for the preparation of the above monoclonal antibody (modified antibody) and its use in antitumor drugs.
  • the present invention is achieved by the following technical measures:
  • the present invention provides a modified antibody which enhances its biological activity by changing the sugar chain composition of the ofatumumab monoclonal antibody.
  • the primary amino acid sequence of the modified antibody is identical to the primary amino acid sequence of the ofatumumab monoclonal antibody, but the sugar chain of the modified antibody and the omalimumab have the expression process.
  • the different sugar chains make the biological activity of the obtained modified antibody significantly enhanced, for example, by 10 to 100 times.
  • the modified antibody enhances antibody-dependent cell-mediated cytotoxicity (ADCC) by reducing the fucose content of the sugar chain of the modified antibody.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the fucose-containing sugar chain accounts for no more than 20% by mass of the total sugar chain of the modified antibody.
  • the sugar chain having fucose accounts for not more than 10% by mass of the total sugar chain of the modified antibody, for example, the mass percentage thereof is Not more than 8%.
  • the sugar chain having fucose accounts for no more than 5% by mass of the total sugar chain of the modified antibody, for example, 0.2. % to 5%.
  • the sugar chain of the modified antibody consisting of carbohydrates does not contain fucose.
  • the present invention provides a method for producing the aforementioned modified antibody, which comprises the step of translating expression in a medium to which an L-fucose analog is added by a eukaryotic host cell capable of expressing an olfaximab.
  • the L-fucose analog comprises 5-alkynyl-L-fucose, 2-deoxy-2-fluoro-L-fucose And 2-fluoro-peracetyl-fucose (2F-Peracetyl-Fucose, commercially available from Merck) and one or more of their corresponding peracylated derivatives and other derivatives.
  • the L-fucose analogue preferably comprises 2-deoxy-2-fluoro-L-fucose and/or a corresponding peracylated derivative And other derivatives.
  • the L-fucose analogue preferably comprises 5-alkynyl-L-fucose and/or corresponding peracylated derivative or other derivative Things.
  • the eukaryotic host cell capable of expressing orfarizumab is preferably a CHO cell line, an SP2/0 cell line or an NSO cell line.
  • the eukaryotic host cell is preferably a CHO cell line.
  • the invention provides a pharmaceutical composition comprising the above modified antibody.
  • the invention provides the use of the modified antibody described in the preparation of an anti-tumor drug.
  • the tumor comprises chronic lymphoma, non-Hodgkin's lymphoma and/or diffuse large B-cell lymphoma.
  • the invention provides the use of the method of preparation in the preparation of an anti-tumor drug.
  • the tumor comprises chronic lymphoma, non-Hodgkin's lymphoma and/or diffuse large B-cell lymphoma.
  • the antitumor drug and its use are preferably chronic lymphoma, non-Hodgkin's lymphoma and diffuse large B cell lymphoma.
  • the invention provides a method of treating a tumor, the method comprising administering to a subject an effective amount of a modified antibody of the invention or a pharmaceutical composition of the invention.
  • the tumor is selected from the group consisting of chronic lymphoma, non-Hodgkin's lymphoma, and/or diffuse large B-cell lymphoma.
  • the main technique for increasing the activity of the target monoclonal antibody ADCC by reducing or removing fucose is to focus on the genetic level of the cell, such as Biowa's POTELLIGENT(R) technology and Roche's GlycoMAb technology.
  • the invention realizes the reduction or removal of the fucose content in the monoclonal antibody of interest by regulating the metabolic pathway of the cell, such as adding a fucose analog during the cultivation, and adjusting the sugar chain composition of the modified antibody by the process control means. , achieved the purpose of significantly increasing the activity of the monoclonal antibody ADCC.
  • Antibodies in mammalian cells require GDP-fucose as a substrate, and ⁇ -1,6 fucose ( ⁇ -1,6 fucose) modification by fucosyltransferase, intracellular GDP- Fucose (GDP-fucose) can be produced from the salvage pathway by utilizing the de novo (synthetic) pathway of glucose or using free fucose. Intracellular GDP-fucose levels produce feedback inhibition of de novo (synthetic) pathway enzymes (such as GMD) or inhibition of fucosyltransferase activity, thereby affecting core fucose modification.
  • GDP-fucose intracellular GDP- Fucose
  • Intracellular GDP-fucose levels produce feedback inhibition of de novo (synthetic) pathway enzymes (such as GMD) or inhibition of fucosyltransferase activity, thereby affecting core fucose modification.
  • fucose analog such as 2F-Peracetyl-Fucose (2F)
  • GDP-2F-fucose GDP-2F-fucose
  • GDP- The synthesis of fucose and inhibition of fucosyltransferase activity, thereby obtaining a low fucose-containing sugar chain antibody, which has higher ADCC activity than orfarizumab.
  • the method is more convenient than genetically engineered cells (such as the patent application method of Glichard Biotech Co., Ltd., CN02818173.5), and the antibody production is also significantly increased.
  • the main advantage of the present invention is that the modified antibody obtained maintains similar safety to orfarizumab while having a higher ADCC activity which would be clinically beneficial in reducing the likelihood of therapeutic resistance to antibodies by the patient.
  • the modified monoclonal antibody prepared according to the invention has significantly higher clinical efficacy than the existing olfamuumab, and has remarkable advancement, and is also in line with the development concept of introduction, digestion and resorption in the development of biomedicine in China. Reduce the risk of drug development.
  • Figure 1 is a MS spectrum of 2F-Peracetyl-Fucose-treated antibody ADCC raised mAb as measured in Example 3.
  • Example 2 is a MS spectrum of an antibody treated with 5-alkynyl-Fucose as measured in Example 3.
  • Figure 3 is a MS map of olfamumab expressed by CHO measured in Example 3.
  • Figure 5 is a schematic representation of the structure of an antibody modified sugar chain.
  • Fig. 6 is a comparison of the antitumor effect of the olfaximab measured in Example 5 before and after the modification of the sugar chain ADCC activity in the animal.
  • Figure 7 is a chromatogram of a representative glycoside measured in Example 9.
  • the present invention demonstrates that the prepared modified antibody is compared with orfarizumab: by reducing the fucose content of the sugar chain in the modified antibody, a modified antibody having higher ADCC activity than the olfaxumab is obtained. It shows stronger anti-tumor activity in the body.
  • the cells used for the resuscitation in the following examples are CHO (DG44)-derived cell line ET-4-66 based on stable expression of orfarizumab (the screening process is described in Peng Zhan et al., and is low for Mongolian and Caucasian species).
  • a cryotube containing ET-4-66 working cells (1.5 ml) was taken from a liquid nitrogen tank, frozen in a 37 °C water bath, and immediately transferred to a 20-30 mL CD FortiCHO medium. In a 125 mL shake flask. The shake flask was placed in a CO 2 incubator and incubated at 37 ° C, 8% CO 2 , 130 rpm.
  • 2F-Peracetyl-Fucose or 5-Alkynyl-L-fucose cultured to 4 ⁇ 10 6 to 5 ⁇ 10 6 cells At a density of /mL, re-seeded in fresh CD FortiCHO medium containing the above fucose analog at 0.8 ⁇ 10 6 to 1.2 ⁇ 10 6 cells/mL for 9 days, and the culture supernatant was purified by affinity chromatography. The fucosose knockout antibody was obtained.
  • a cryotube containing ET-4-66 working cells (1.5 ml) was taken from a liquid nitrogen tank, frozen in a 37 °C water bath, and immediately transferred to a 20-30 mL FortiCHO (addition of MTX500nM). The medium was in a 125 mL shake flask. The shake flask was placed in a CO 2 incubator, and cultured at 37 ° C, 8% CO 2 , 130 rpm for 2 to 3 days.
  • Shake flask expansion When the cell density is increased to 1.5 ⁇ 10 6 to 3.5 ⁇ 10 6 cells / mL, the cells are expanded. When necessary, an appropriate amount of fresh FortiCHO medium is added as needed, and the density of the cells after expansion is 0.4 ⁇ 10 6 to 0.6 ⁇ 10 6 cells/mL. The shake flask is replaced according to the volume of the cell fluid, and the culture volume does not exceed 1/3 of the total volume of the shake flask. The N-1 generation was started by adding 2F-Peracetyl-Fucose at a final concentration of 50 ⁇ M, and culture (N refers to the cell culture generation for production) for the reactor experiment.
  • pH set 7.0 ⁇ 0.05, controlled by CO 2 and 0.5M NaHCO 3 PID feedback;
  • DO set 40% (100% oxygen in air), controlled by oxygen PID feedback
  • Rotation speed control 80-250 rpm, initial rotation speed 80 rpm, 1 hr after inoculation, the rotation speed is increased to 150 rpm, the rotation speed is increased by 200 rpm on the 5th day, and if the Sparger ventilation exceeds 100 ml/min, the rotation speed can be increased to 250 rpm.
  • the cultured cells are stopped, and the supernatant is centrifuged to purify.
  • the main steps of purification include depth filtration, affinity chromatography, acid incubation inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration and ultrafiltration steps.
  • the purified ADCC-promoting antibody was subjected to an in vivo animal efficacy test according to Example 5, and the ADCC-promoting antibody was measured in the same manner as in Example 9 to determine that the total fucose-free glycoform was 97.8%, and the fucose-containing sugar was contained. The total amount is 2.2%.
  • Figures 1 to 4 are the fucose knockouts prepared in Example 1 based on the QSTAR XL (AB Sciex) mass spectrometer.
  • the monoclonal antibody and the original research product Olfazumab (GSK, batch c669907) were tested for glycan profiles.
  • the specific analysis of the MS spectrum of the monoclonal antibody (antibody obtained without the addition of fucose analog) (Fig. 3) and the original olfamumab (Fig. 4) is shown in Table 2.
  • each modified sugar chain in Table 2 is shown in Figure 5. From the results of MASS in Table 2, it is known that 2F-Peracetyl-Fucose and 5-- are added during the culture process compared to the original and the untreated antibody. 5-Alkynyl-L-fucose completely alters the modified sugar chain of the antibody. The molecular weight of each major signal peak on the mass spectrum is based on the main peak shape of the original product, and the molecular weight of 292 Da (the molecular weight of two fucose residues) is subtracted.
  • the ADCC effect assay was performed based on engineered NK cells to determine the killing of Wil2-s cells by antibodies.
  • Wil2-s target cell preparation Take the target cell Wil2-s in logarithmic growth phase, centrifuge at 1000 rpm for 5 minutes, wash once with RPMI 1640 (#11835), adjust the cell concentration to 5 ⁇ 10 5 /ml, in 96 wells. 50 ⁇ l per well in a round bottom plate (except for effector cell autofluorescence control wells and blank background control wells);
  • Example 2 Dilute the original drug or other sample to be tested (sample prepared in Example 1) to 1 ⁇ g/ml with RPMI1640, then do a 5-fold gradient dilution, dilute 9 points, and add a blank spot containing no antibody; 96-well plate Add 50 ⁇ l per well to the target cells, and make 2 replicate wells per concentration;
  • NK-92MI/CD16a effector cells Take effector cells NK-92MI/CD16a in logarithmic growth phase, centrifuge at 1000 rpm for 5 minutes, wash once with RPMI 1640, adjust the cell concentration to 5 ⁇ 10 5 /ml, in a 96-well round bottom plate. Medium (except for target cell autofluorescence control wells and target cells maximal lysate control wells) 100 ⁇ l per well;
  • 96-well plates were incubated for 4 hours at 37 ° C, 5% CO 2 ;
  • a) 96-well plate is placed at room temperature after incubation, and equilibrated for 20 to 30 minutes; all detection reagents are first equilibrated to room temperature;
  • the X-axis data is the mass concentration (ng/ml) of the log-transformed original drug, and the Y-axis data is ADCC%.
  • the metabolic flux of the cell strain was changed by adding the fucose substitutes 2F-Peracetyl-Fucose and 5-alkynyl-L-fucose, thereby preparing A modified antibody with high ADCC activity
  • the ADCC activity of the modified antibody (EC 50 is 0.1718-0.3372 ng/mL) is similar to that of Roche's newly approved ADCC-enhanced Gazyva mAb (EC 50 is 0.3836 ng/mL), which is much higher than the original one.
  • ADCC activity EC 50 of 2.718 ng/mL and 3.097 ng/mL, respectively) of orofaizumab and CHO-expressed orfarizumab.
  • the present invention is supported by the regulation of cell metabolism by the addition of a fucose analog to obtain an antibody having a low fucose content.
  • mice BALB/cA-nude nude mice, 6-7 weeks old, sputum, nude mice were subcutaneously inoculated with human B cell lymphoma Daudi cells, and after the tumors were grown to 100-150 mm 3 , the animals were randomly grouped (D0).
  • the dosage and administration schedule are shown in Table 5.
  • the tumor volume was measured 2 to 3 times a week, the rats were weighed, and the data were recorded.
  • the tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width, respectively.
  • T/C (%) (TT 0 ) / (CC 0 ) ⁇ 100% where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the start of the experiment.
  • D0 first dosing time
  • P value means compared to solvent
  • the equifacilizumab and ADCC-enhanced opfazumab significantly inhibited CD20-positive B cells.
  • the growth of subcutaneous xenografts of lymphoma in Daudi nude mice was 84% and 95%, respectively, and 2/6 tumors were partially resolved in the olfaximab group; 2/6 tumor partial regression and 1 in the ADCC boosting antibody group.
  • Example 6 2-Fluoro-peracetyl-fucose (2F-Peracetyl-Fucose, commercially available from Merck) for the preparation of high ADCC active antibodies in the first batch of 50 L reactor culture
  • a cell preserved in a cryotube was taken from a liquid nitrogen tank (loading 1.5 mL), frozen in a 37 ° C water bath, and immediately transferred to a 125 mL shake flask containing 20-30 mL CD FortiCHO medium. The shake flask was placed in a CO 2 incubator, and cultured at 37 ° C, 8% CO 2 , humidity 80%, and 130 rpm for 3 to 4 days.
  • Shake flask expansion When the cell density is increased to 2 ⁇ 10 6 to 4 ⁇ 10 6 cells / mL, the cells are expanded. When necessary, an appropriate amount of fresh CD Dynamis medium was added as needed, and the density of the cells after expansion was 0.4 ⁇ 10 6 to 0.6 ⁇ 10 6 cells/mL. Replace the appropriate volume of the shake flask according to the volume of the cell fluid. The culture volume should not exceed 1/3 of the total volume of the shake flask.
  • the reactor is Applikon EZ Control 15L glass jar, parameter control: (1) pH control: 7.0 ⁇ 0.1, controlled by CO 2 and 0.5M NaHCO 3 PID feedback; (2) DO Control: set 40 ⁇ 10%, with pure oxygen PID feedback control; (3) speed control: 80 ⁇ 200 rpm, starting speed 80 rpm, 1 hr after inoculation, the speed is increased to 120 rpm.
  • Temperature control 36.5 °C ⁇ 0.5 °C;
  • Gas control Overlay compressed air, constant speed 50 ⁇ 100mL / min;
  • Sparger CO 2 PID feedback control CO 2 set upper limit 300mL / min,
  • Sparger O 2 PID Feedback control O 2 sets the upper limit of 300mL / min. Inoculate 5.5-6.5L, the density after inoculation is controlled at 0.5 ⁇ 10 6 cells/mL, culture for 4-5 days, density to 4 ⁇ 6 ⁇ 10 6 cells/mL, enter the 50L SUB or 50L Mobius reactor. Enlarge the culture.
  • 50L SUB reactor operation Prepare Applikon controller and 50L SUB bag and matching pipeline (all sterile) before inoculation, prepare 20L CD Dynamis medium and filter into bag, the medium contains 2-fluoro- Peroxy-fucose (2F-Peracetyl-Fucose) 50 ⁇ 200 ⁇ M, temperature control is 36.5 ⁇ 0.5 ° C, surface ventilation 50 ⁇ 100mL / min, deep Sparger air is 100mL / min, speed 80rpm, overnight calibration of dissolved oxygen 100%.
  • 2F-Peracetyl-Fucose 2-fluoro- Peroxy-fucose
  • the training parameters were set: DO 40 ⁇ 10%, pH 7.0 ⁇ 0.1, temperature 36.5 ⁇ 0.5°C, and after inoculation for 30 minutes, the cell density and viability were sampled and measured, and the rotation speed was 80-100 rpm. EFC and glucose were added during the cultivation.
  • the culture period is 14 to 16 days, the survival rate is about 70%, the culture is finished, and the fermentation liquid is harvested.
  • the main steps of the fermentation broth purification include depth filtration, affinity chromatography, acid incubation inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration and ultrafiltration steps.
  • the purified sample was sent for glycan analysis.
  • the initial concentration of L-glutamine is 6 mM, and the glucose is controlled by 2 to 4 g/L.
  • the density after cell inoculation is controlled at 0.8-1.2 ⁇ 10 6 cells/mL;
  • Tyr was added to the basal medium on day 0 in an amount of 1/500 (working volume) and Tyr was prepared at a concentration of 100 g/L.
  • Example 7 5-Alkynyl-Fucose (CAS No.: 1193251-61-4) was prepared in a second batch of 50 L reactor to prepare high ADCC active antibody.
  • a cell preserved in a cryotube was taken from a liquid nitrogen tank (loading 1.5 mL), frozen in a 37 ° C water bath, and immediately transferred to a 125 mL shake flask containing 20-30 mL CD FortiCHO medium. The shake flask was placed in a CO 2 incubator, and cultured at 37 ° C, 8% CO 2 , humidity 80%, and 130 rpm for 3 to 4 days.
  • Shake flask expansion When the cell density is increased to 2 ⁇ 10 6 to 4 ⁇ 10 6 cells / mL, the cells are expanded. When necessary, an appropriate amount of fresh CD FortiCHO medium was added as needed, and the density of the cells after expansion was 0.4 ⁇ 10 6 to 0.6 ⁇ 10 6 cells/mL. Replace the appropriate volume of the shake flask according to the volume of the cell fluid. The culture volume should not exceed 1/3 of the total volume of the shake flask.
  • the reactor is Applikon EZ Control 15L glass jar, parameter control: (1) pH control: 7.0 ⁇ 0.1, controlled by CO 2 and 0.5M NaHCO 3 PID feedback; (2) DO Control: set 40 ⁇ 10%, with pure oxygen PID feedback control; (3) speed control: 80 ⁇ 200 rpm, starting speed 80 rpm, 1 hr after inoculation, the speed is increased to 120 rpm.
  • Temperature control 36.5 °C ⁇ 0.5 °C;
  • Gas control Overlay compressed air, constant speed 50 ⁇ 100mL / min;
  • Sparger CO 2 PID feedback control CO 2 set upper limit 300mL / min,
  • Sparger O 2 PID Feedback control O 2 sets the upper limit of 300mL / min. Inoculate 5.5-6.5L, the density is controlled at 0.5 ⁇ 10 6 cells/mL after inoculation, culture for 4 to 5 days, and the density reaches 4 ⁇ 10 6 to 6 ⁇ 10 6 cells/mL, and enter 50L SUB or 50L Mobius reaction. The test was scaled up.
  • 50L Mobius reactor operation Prepare Applikon controller and 50L SUB bag and matching pipeline (all sterile) before inoculation, prepare 20L CD FortiCHO medium and filter into bag, 5-alkynyl group -L-fucose (5-Alkynyl-Fucose) 50 ⁇ 200 ⁇ M, temperature control is 36.5 ⁇ 0.5 ° C, surface ventilation 50-100mL / min, deep Sparger air is 100mL / min, speed 80rpm, overnight calibration dissolved oxygen is 100 %.
  • Culture parameter setting DO 40 ⁇ 10%, pH 7.0 ⁇ 0.1, temperature 36.5 ⁇ 0.5°C, after inoculation for 30 min, samples were taken to measure cell density and viability, and the rotation speed was 80-100 rpm. EFC and Gluc were added during the cultivation. The culture period is 14 to 16 days, the survival rate is about 70%, the culture is finished, and the fermentation liquid is harvested.
  • the main steps of the fermentation broth purification include depth filtration, affinity chromatography, acid incubation inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration and ultrafiltration steps. The purified sample was sent for glycan analysis.
  • 5-Alkynyl-L-fucose was added and controlled in the second batch of 50L Mobius reactor in a secondary batch culture according to the feeding strategy and culture conditions designed according to Table 7:
  • the initial concentration of L-glutamine is 6 mM, and the glucose is controlled by 2 to 4 g/L.
  • the density after cell inoculation is controlled at 0.8 ⁇ 10 6 to 1.2 ⁇ 10 6 cells/mL;
  • Tyr was added to the basal medium on day 0 in an amount of 1/500 (working volume) and Tyr was prepared at a concentration of 100 g/L.
  • Example 8 2-Deoxy-2-fluoro-L-Fucose (CAS No.: 70763-62-1) was prepared in a third batch of 50 L reactor to prepare a high ADCC. Active antibody
  • a cell preserved in a cryotube was taken from a liquid nitrogen tank (loading 1.5 mL), frozen in a 37 ° C water bath, and immediately transferred to a 125 mL shake flask containing 20-30 mL CD FortiCHO medium. The shake flask was placed in a CO 2 incubator, and cultured at 37 ° C, 8% CO 2 , humidity 80%, and 130 rpm for 3 to 4 days.
  • Shake flask expansion When the cell density is increased to 2 ⁇ 10 6 ⁇ 4 cells / mL, the cells are expanded. During the operation, an appropriate amount of fresh CD FortiCHO medium was added as needed, and the density of the cells after expansion was 0.4 ⁇ 10 6 to 0.6 cells/mL. Replace the appropriate volume of the shake flask according to the volume of the cell fluid. The culture volume should not exceed 1/3 of the total volume of the shake flask.
  • the reactor is Applikon EZ Control 15L glass jar, parameter control: (1) pH control: 7.0 ⁇ 0.1, controlled by CO 2 and 0.5M NaHCO 3 PID feedback; (2) DO Control: set 40 ⁇ 10%, with pure oxygen PID feedback control; (3) speed control: 80 ⁇ 200 rpm, starting speed 80 rpm, 1 hr after inoculation, the speed is increased to 120 rpm.
  • Temperature control 36.5 °C ⁇ 0.5 °C;
  • Gas control Overlay compressed air, constant speed 50 ⁇ 100mL / min;
  • Sparger CO 2 PID feedback control CO 2 set upper limit 300mL / min,
  • Sparger O 2 PID Feedback control O 2 sets the upper limit of 300mL / min. Inoculate 5.5-6.5L, the density is controlled at 0.5 ⁇ 10 6 cells/mL after inoculation, culture for 4 to 5 days, and the density reaches 4 ⁇ 10 6 to 6 ⁇ 10 6 cells/mL, and enter 50L SUB or 50L Mobius reaction. The test was scaled up.
  • 50L Mobius reactor operation Prepare Applikon controller and 50L SUB bag and matching pipeline (all sterile) before inoculation, prepare 20L CD Dynamis medium and filter into bag, 2-deoxy-containing medium 2-Deoxy-2-fluoro-L-Fucose 50-200 ⁇ M, temperature control is 36.5 ⁇ 0.5°C, surface aeration is 50-100mL/min, and deep Sparger air is 100mL/min. The rotational speed was 80 rpm, and the dissolved oxygen was corrected to 100% overnight.
  • Culture parameter setting DO 40 ⁇ 10%, pH 7.0 ⁇ 0.1, temperature 36.5 ⁇ 0.5°C, after inoculation for 30 min, samples were taken to measure cell density and viability, and the rotation speed was 80-100 rpm. EFC and Gluc were added during the cultivation.
  • the culture period is 14 to 16 days, the survival rate is about 70%, the culture is finished, and the fermentation liquid is harvested.
  • the main steps of the fermentation broth purification include depth filtration, affinity chromatography, acid incubation inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration and ultrafiltration steps.
  • the purified sample is sent for glycoform analysis, ADCC activity assay, and the like.
  • the initial concentration of L-glutamine is 6 mM, and the glucose is controlled by 2 to 4 g/L.
  • the density after cell inoculation is controlled at 0.8-1.2 ⁇ 10 6 cells/mL;
  • Tyr was added to the basal medium on day 0 in an amount of 1/500 (working volume) and Tyr was prepared at a concentration of 100 g/L.
  • 500 ⁇ g of the sample proteins obtained in Examples 6 to 8 were added to 1 ⁇ PBS to 500 ⁇ L (1 ⁇ g/ ⁇ L) for mixing; 5 ⁇ L of PNGaseF enzyme was added, and the mixture was mixed and reacted in a water bath at 37 ° C for 3 hours.
  • glycoside standard Glycan Test waters, P.N.186006349, S.N.W19011504, 250 ⁇ L 60% acetonitrile reconstituted
  • 2-AB labeled samples were analyzed by high performance liquid chromatography with a fluorescence detector.
  • the chromatographic conditions are shown in Table 9 below:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了岩藻糖含量降低的奥法木抗体，其ADCC (抗体依赖性细胞介导的细胞毒性) 活性显著性提升。还提供了制备所述抗体的方法，其中在培养过程中添加岩藻糖类似物。

Description

抗体依赖性细胞介导的细胞毒性增强的奥法木抗体 技术领域
本发明属于基因工程领域，涉及一个通过改变已上市的奥法木(Ofatumumab，Arzerra)单抗的糖链组成，获得抗体依赖性细胞介导的细胞毒性(ADCC)显著性增强的修饰抗体。本发明还涵盖了该修饰抗体的制备方法及其在治疗肿瘤，尤其是CD20高表达的慢性淋巴瘤，非霍奇金氏淋巴瘤和弥漫性大B细胞淋巴瘤中的应用。
背景技术
CD20(亦称为人B淋巴细胞限制性分化抗原)是前B细胞核成熟的B淋巴细胞上的疏水性跨膜蛋白(Valentine et al.,J.Bio.Chem.,1989,V264:11282-7)。研究发现，超过90％的非霍奇金氏淋巴瘤患者、慢性淋巴瘤患者及大B细胞淋巴瘤患者B细胞表面高度表达CD20分子(Anderson et al.,Blood,1984,V63:1424-33)，但造血干细胞、原B细胞、正常浆细胞及其它组织中并未发现前述特征(Tedder et al.,Immunology,1985,V135:973-9)。基于此特征，Biogen公司于1997年上市了特异性针对CD20的人鼠嵌合型抗体Rituxan(Rituximab)，用于治疗慢性淋巴瘤、非霍奇金氏淋巴瘤等，并在临床上成为治疗的金标准。Genmab公司随后在2009年上市了针对CD20大小环区域的全人抗体-奥法木单抗(ofatumumab)，该抗体的补体依赖性细胞毒性(CDC)远高于Rituxan，已被FDA批准用于慢性B细胞淋巴瘤的一线治疗，目前该品种正在进行非霍奇金氏淋巴瘤一线治疗的临床研究。
Herter等对奥法木单抗(ofatumumab)与Rituxan开展的头对头对比研究表明，奥法木单抗在体外和动物体内的各项活性指标均不逊于Rituxan，且其补体依赖性细胞毒性(CDC)远高于Rituxan(Herter et al.,Mol.Cancer.Ther.2013；12:2031-42)。针对已取得良好疗效的奥法木单抗，为使其在临床上发挥更佳的临床效果，仍存在进一步改良该抗体的技术需求。
发明内容
本发明目的之一是通过调节细胞代谢流的方式改变奥法木(ofatumumab)单抗的糖链组成提升其生物学活性，尤其是抗体依赖性细胞介导的细胞毒性(ADCC)活性，获得一个具有更高生物学活性的新抗体(修饰抗体)。其抗肿瘤活性的提升在本发明中已通过细 胞水平的体外试验及动物水平的体内药效试验证实。
同时，优选CHO表达系统替代NS0表达系统，从而降低或消除NS0系统表达奥法木单抗时含有的Di-α1,3GalGal糖修饰的组分，使本发明单抗具有更低的免疫原性。
本发明的另一个目的是提供上述单抗(修饰抗体)的制备方法及其在抗肿瘤药物中的应用。
本发明是通过下列技术措施实现的：
本发明提供一种通过改变奥法木(ofatumumab)单抗的糖链组成提升其生物学活性的修饰抗体。
根据本发明的具体实施方式，所述修饰抗体的一级氨基酸序列与奥法木(ofatumumab)单抗一级氨基酸序列一致，但其在表达过程中修饰抗体的糖链与奥法木单抗具有的糖链不同，使获得的修饰抗体的生物学活性较奥法木抗体显著增强，例如提升10～100倍。
根据本发明的具体实施方式，所述修饰抗体是通过降低修饰抗体的糖链中岩藻糖含量，增强抗体依赖性细胞介导的细胞毒性(ADCC)。
根据本发明的具体实施方式，在由碳水化合物所组成的修饰抗体的糖链中，具岩藻糖的糖链占修饰抗体的总糖链的质量百分比不高于20％。
根据本发明的具体实施方式，在由碳水化合物所组成的修饰抗体的糖链中，具岩藻糖的糖链占修饰抗体的总糖链的质量百分比不高于10％，例如其质量百分比为不高于8％。
根据本发明的具体实施方式，进一步地，在由碳水化合物所组成的修饰抗体的糖链中，具岩藻糖的糖链占修饰抗体的总糖链的质量百分比不高于5％，例如0.2％～5％。
根据本发明的具体实施方式，进一步地，由碳水化合物所组成的修饰抗体的糖链中，不含岩藻糖。
本发明提供前述修饰抗体的一种制备方法，所述方法包括由能表达奥法木单抗的真核宿主细胞在添加了L-岩藻糖类似物的培养基中翻译表达的步骤。
根据本发明的具体实施方式，在本发明所述方法中，所述L-岩藻糖类似物包括5-炔基-L-岩藻糖、2-脱氧-2-氟-L-岩藻糖、2-氟-过乙酰基-岩藻糖(2F-Peracetyl-Fucose，可为Merck公司目录商品)和它们相应的过酰化衍生物及其他的衍生物中的一种或多种。
根据本发明的具体实施方式，在本发明所述方法中，所述L-岩藻糖类似物优选包括2-脱氧-2-氟-L-岩藻糖和/或相应的过酰化衍生物及其他衍生物。
根据本发明的具体实施方式，在本发明所述方法中，所述L-岩藻糖类似物优选包括5-炔基-L-岩藻糖和/或相应的过酰化衍生物或其他衍生物。
根据本发明的具体实施方式，在本发明所述方法中，所述能表达奥法木单抗的真核宿主细胞优选CHO细胞系、SP2/0细胞系或NS0细胞系。
根据本发明的具体实施方式，在本发明所述方法中，所述真核宿主细胞优选CHO细胞系。
另一方面，本发明提供一种药物组合物，所述组合物包含上述修饰抗体。
另一方面，本发明提供所述的修饰抗体在制备抗肿瘤药物中的应用。优选地，所述肿瘤包括慢性淋巴瘤、非霍奇金氏淋巴瘤和/或弥漫性大B细胞淋巴瘤。
另一方面，本发明提供所述制备方法在制备抗肿瘤药物中的应用。优选地，所述肿瘤包括慢性淋巴瘤、非霍奇金氏淋巴瘤和/或弥漫性大B细胞淋巴瘤。
所述的抗肿瘤药物及其应用，优选慢性淋巴瘤，非霍奇金氏淋巴瘤和弥漫性大B细胞淋巴瘤。
另一方面，本发明提供一种治疗肿瘤的方法，所述方法包括给予受试体有效剂量的本发明所述的修饰抗体或本发明所述的药物组合物。所述肿瘤选自慢性淋巴瘤、非霍奇金氏淋巴瘤和/或弥漫性大B细胞淋巴瘤。
以下主要提供本发明的主要步骤：
通过降低或去除岩藻糖提升目的单抗ADCC活性的主要技术是集中在细胞基因水平层面上进行改造，如Biowa公司的POTELLIGENT(R)技术和罗氏的GlycoMAb技术。本发明突破性的通过调节细胞的代谢途径而实现了降低或去除目的单抗中岩藻糖含量，如培养过程中添加岩藻糖类似物，通过工艺控制手段对修饰抗体的糖链组成进行调节，达到了显著性提升单抗ADCC活性的目的。
抗体在哺乳动物细胞中需要以GDP-岩藻糖(GDP-fucose)为底物，通过岩藻糖转移酶进行α-1,6岩藻糖(α-1,6fucose)修饰，细胞内GDP-岩藻糖(GDP-fucose)可以通过利用葡萄糖从头(合成)途径或者利用游离的岩藻糖从补救途径生成。胞内的GDP-岩藻糖(GDP-fucose)水平会对从头(合成)途径的酶(如GMD)产生反馈抑制，或者抑制岩藻糖转移酶的活性，从而影响核心岩藻糖修饰。通过在培养过程中添加岩藻糖类似物，如2F-Peracetyl-Fucose(简称2F)，形成GDP-2F-岩藻糖(GDP-2F-fucose)，抑制正常的GDP-岩藻糖(GDP-fucose)的合成，并且抑制岩藻糖转移酶活性，从而获得低岩藻糖含量的糖链抗体，该修饰抗体较奥法木单抗具有更高的ADCC活性。同时，该法较通过基因工程化改造细胞(如格黎卡特生物技术股份公司申请专利方法，CN02818173.5)更加便捷，抗体产量也显著增加。
本发明的有益效果：
本发明主要的优点：获得的修饰抗体保持与奥法木单抗相似的安全性同时，其ADCC活性更高，这种改变在临床上将有益于降低因患者对抗体发生治疗性抵制的几率。基于本发明所制备的修饰单抗，在临床的药效显著高于现有的奥法木单抗，具有显著的先进性，也符合我国生物医药开发方面的引进、消化再吸收的开发理念，降低了药物开发的风险。
附图说明
图1为实施例3测得的2F-Peracetyl-Fucose处理的抗体ADCC提升单抗的MS图谱。
图2为实施例3测得的5-炔基-L-岩藻糖(5-Alkynyl-Fucose)处理的抗体的MS图谱。
图3为实施例3测得的CHO表达的奥法木单抗MS图谱。
图4为实施例3测得的原研奥法木单抗MS图谱。
图5为抗体修饰糖链结构的示意图。
图6为实施例5测得的奥法木单抗通过修饰糖链ADCC活性改变前后其动物体内抑瘤效果比较。
图7为实施例9测得的代表性糖苷的色谱图。
具体实施方式
下面结合具体实施例及附图对本发明作进一步阐述，但不限制本发明。在此，本发明证明了所制备的修饰抗体与奥法木单抗相比：通过降低修饰抗体中糖链的岩藻糖含量，获得较奥法木单抗具有更高的ADCC活性的修饰抗体，在体内出示了更强的抗肿瘤活性。
以下实施例中复苏所采用的细胞为基于稳定表达奥法木单抗的CHO(DG44)来源的细胞株ET-4-66(其筛选过程参见于鹏展等，对蒙古人和高加索人种低免疫原性的抗CD20的人抗体，申请号CN104341503A，其全部内容在此文引作参考)。
实施例1用不同岩藻糖类似物在不同发酵规模(3L)条件下制备岩藻糖敲除的抗体
复苏：从液氮罐中取存有ET-4-66工作细胞的冻存管一支(装量1.5ml)，37℃水浴化冻，立即将种子液转入含有20～30mL CD FortiCHO培养基的125mL摇瓶中。将摇瓶放置于CO2培养箱中，37℃，8％CO2，130rpm培养。
接种：取对数生长期的细胞按照0.8×106～1.2×106cells/mL细胞密度接种于125mL摇瓶中，培养体积30mL，在培养基中分别添加终浓度分别为200μM、100μM、50μM的2F-过乙酰基-岩藻糖(2F-Peracetyl-Fucose)或5-炔基-L-岩藻糖(5-Alkynyl-L-fucose)，培养至4×106～5×106cells/mL密度时，按0.8×106～1.2×106cells/mL重新接种于含上述岩藻糖类似 物的新鲜的CD FortiCHO培养基中连续培养9天，培养上清经亲和层析纯化得到岩藻糖敲除的抗体。
实施例2高ADCC活性抗体制备
复苏：从液氮罐中取存有ET-4-66工作细胞的冻存管一支(装量1.5ml)，37℃水浴化冻，立即将种子液转入含有20～30mL FortiCHO(添加MTX500nM)培养基的125mL摇瓶中。将摇瓶放置于CO2培养箱中，37℃，8％CO2，130rpm培养2～3天。
摇瓶扩种：当细胞密度增加至1.5×106～3.5×106cells/mL时，对细胞进行扩种。操作时根据需要，补充适量的新鲜FortiCHO培养基，扩种后细胞的密度在0.4×106～0.6×106cells/mL。根据细胞液体积更换摇瓶，培养体积不超过摇瓶总体积的1/3。N-1代开始添加终浓度为50μM的2F-Peracetyl-Fucose，培养(N指生产用的细胞培养代次)，用于反应器实验。
以0.8×106～1.2×106cells/mL细胞密度接种，根据实验方案中(见表1)设计的补料策略和培养条件进行添加和控制：
表1 3L反应器性能测试实验设计
培养基和补料 2F/％(V/V) 细胞代次 备注 工作体积
CD FortiCHO+EFC+Tyr/Cys 0.5 P14 WCB 1100mL
备注：葡萄糖控制＞2.5g/L；
EFC：第3天开始每天按初始工作体积在线补加3％(V/V)，补加总量为33-36％；
Tyr：第7天添加1/500(V/V)，第13天添加1/1000(V/V)；
Cys：第7天添加1/100(V/V)，第13天添加1/200(V/V)。反应器参数控制如下：
工作体积：起始1.2L培养基；
温度：控制在36.5℃；
pH：设定7.0±0.05，分别用CO2和0.5M NaHCO3PID反馈控制；
DO：设定40％(以空气中氧气为100％)，用氧气PID反馈控制；
转速：控制80～250rpm，起始转速80rpm，接种后1hr，转速提高至150rpm，第5天转速提高200rpm，若Sparger通气量超过100ml/min，转速可提高至250rpm。
气体控制：Overlay压缩空气40-150ml/min；
Sparger CO2PID反馈控制pH设定上限300ml/min；
O2PID反馈控制DO设定上限300ml/min；
每天取样，观察细胞生长状态，计算活细胞数量和细胞活率，用Nova进行生化分析， 并移取大于1ml样品在1000rpm，3min条件下离心，取上清-80℃保存。
当细胞活率低于70％，或者活细胞数是峰值的一半时，停止培养收获细胞，离心留取上清纯化。纯化主要步骤包括深层过滤、亲和层析、酸孵育灭活、阳离子交换层析、阴离子交换层析、纳滤及超滤步骤。纯化后的ADCC提升抗体按实施例5进行动物体内药效实验，该ADCC提升抗体按照实施例9相同的方法，测得不含岩藻糖糖型总量为97.8％，及含岩藻糖糖型总量为2.2％。
实施例3本发明所述抗体的糖型分析
糖型的解析主要基于ESI-QTOF对抗体重链以结合形式存在的糖链进行分析，图1至图4是基于QSTAR XL(AB Sciex)质谱仪分别对实施例1制备得到的岩藻糖敲除的单抗和原研品奥法木单抗(GSK，批次c669907)的测试糖谱。培养过程中添加2F-Peracetyl-Fucose获得的抗体(图1)、添加5-炔基-L-岩藻糖(5-Alkynyl-L-fucose)获得的抗体(图2)、CHO表达的奥法木单抗(未添加岩藻糖类似物获得的抗体)(图3)与原研奥法木单抗(图4)其MS图谱具体解析见表2。
表2奥法木单抗与ADCC提升样品的MS信号峰解析
Figure PCTCN2016089820-appb-000001
Figure PCTCN2016089820-appb-000002
表2中各修饰糖链的结构示意图如图5所示，从表2的MASS结果可知，相比于奥法木原研品和未处理抗体，培养过程中通过添加2F-Peracetyl-Fucose和5-炔基-L-岩藻糖(5-Alkynyl-L-fucose)完全改变了抗体的修饰糖链。在质谱图上表现为各主要信号峰的分子量为在原研品的主要峰型基础上，都减去了292Da的分子量(2个岩藻糖残基分子量)。通过确证，确定皆脱掉了2个岩藻糖(由于单抗是2个相同重链通过链间二硫键连结在一起)，所以培养过程中，添加2F-Peracetyl-Fucose和5-炔基-L-岩藻糖(5-Alkynyl-L-fucose)均能有效移除岩藻糖。
按照下文实施例9相同的方法，测得不含岩藻糖糖型总量以及含岩藻糖糖型总量如表3所示：
表3
Figure PCTCN2016089820-appb-000003
实施例4本发明所述抗体ADCC活性的比较分析
ADCC效应测定是基于工程化改造的NK细胞进行，测定抗体对Wil2-s细胞的杀伤。
Wil2-s靶细胞准备：取处于对数生长期的靶细胞Wil2-s，1000rpm，离心5分钟，用RPMI 1640(#11835)洗涤一次，调整细胞浓度为5×105/ml，在96孔圆底板中(除了效应细胞自发荧光对照孔和空白背景对照孔外)每孔加入50μl；
用RPMI1640将原研药或其他待检测样品(实施例1所制得的样品)稀释至1μg/ml，然后做5倍梯度稀释，稀释9个点，另外增加一个不含抗体的空白点；96孔板每孔加入50μl与靶细胞混合，每个浓度做2个复孔；
96孔板在37℃，5％CO2孵育30分钟；
NK-92MI/CD16a效应细胞：取对数生长期的效应细胞NK-92MI/CD16a，1000rpm，离心5分钟，用RPMI 1640洗涤一次，调整细胞浓度为5×105/ml，在96孔圆底板中(除了靶细胞自发荧光对照孔和靶细胞最大裂解对照孔外)每孔加入100μl；
96孔板在37℃，5％CO2孵育4小时；
用CytoTox-ONE试剂盒检测荧光信号：
a)96孔板孵育结束后放置室温，平衡20～30分钟；所有检测试剂均先平衡至室温；
b)靶细胞最大裂解孔内每孔加入4μl裂解液；
c)96孔板1000rpm，离心5分钟；
d)取上清100μl，移至96孔黑板(#3904)；
e)每孔加入100μl反应底物，室温放置10分钟；
f)每孔加入50μl反应终止液，混合均匀。
在酶标仪SpectraMax M5记录Fluorescence Ex560nm/Em590nm信号。
数据处理：a)所有值应先减去空白对照组值；按下面公式计算杀伤率ADCC％＝[(实验组-靶细胞自发释放组-效应细胞自发释放组)/(靶细胞最大释放组-靶细胞自发释放
组)]×100％
b)X轴数据为log转换的原研药的质量浓度(ng/ml)，Y轴数据为ADCC％。
c)用GraphPad Prism 5作非线性回归，使用4参数公式f(x)＝B+(A-B)/(1+10^((C-x)×D))拟合获得测试样品杀死Wil2-s细胞的回归曲线。计算EC50，R2等，所得结果如表4所示。
表4.细胞水平测定的ADCC结果
Figure PCTCN2016089820-appb-000004
从表4可以看出，通过添加岩藻糖替代物2F-Peracetyl-Fucose和5-炔基-L-岩藻糖 (5-Alkynyl-L-fucose)，改变细胞株的代谢流，从而制备出高ADCC活性的修饰抗体，该修饰抗体的ADCC活性(EC50为0.1718～0.3372ng/mL)与罗氏新批准的ADCC提升的Gazyva单抗相似(EC50为0.3836ng/mL)，远高于原研奥法木单抗和CHO表达的奥法木单抗的ADCC活性(EC50分别为2.718ng/mL和3.097ng/mL)。从而支持了本发明所述：通过添加岩藻糖类似物调控细胞的代谢，从而获得低岩藻糖含量的抗体。
实施例5本发明所述抗体体内活性的评价
BALB/cA-nude裸小鼠，6～7周，♀，裸小鼠皮下接种人B细胞淋巴瘤Daudi细胞，待肿瘤生长至100～150mm3后，将动物随机分组(D0)。给药剂量和给药方案见表5。每周测2～3次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)×100％其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
给药方案及实验结果见表5及图6。
表5.各抗体对人B细胞淋巴瘤Daudi裸小鼠皮下移植瘤的疗效
Figure PCTCN2016089820-appb-000005
注：D0:第一次给药时间；P值指与溶剂相比；采用Student’s t检验。实验开始时小鼠数目：溶剂组n＝12，治疗组n＝6。
从表5及图6中可以看出，奥法木单抗、ADCC提升的奥法木单抗(3mg/kg，静脉给药IV，每周2次，共4次)明显抑制CD20阳性B细胞淋巴瘤Daudi裸小鼠皮下移植瘤的生长，抑瘤率分别为84％和95％,奥法木单抗组有2/6肿瘤部分消退；ADCC提升抗体组有2/6肿瘤部分消退和1/6肿瘤完全消退；对照药物Gazyva相同剂量和方案对Daudi的抑瘤率为89％，有1/6肿瘤部分消退和1/6肿瘤完全消退；另一个参比药物美罗华
Figure PCTCN2016089820-appb-000006
(30mg/kg， IV，每周2次，共4次)对Daudi的抑瘤率为109％，有1/6肿瘤部分消退和2/6肿瘤完全消退。荷瘤小鼠对以上药物均能很好耐受，没有体重减轻等症状发生。ADCC提升抗体对Daudi的疗效强于奥法木，与Gazyva相似。
为证实该发明可用于不同规模的修饰抗体制备及发明中所述岩藻糖类似物调节代谢流以获得高ADCC活性的修饰抗体，在实施例6～8中分别采用前述3种L-岩藻糖衍生物在50L规模发酵体积上进行放大研究，结果表明：本发明的工艺可重复性高。
实施例6：2-氟-过乙酰基-岩藻糖(2F-Peracetyl-Fucose，Merck公司市售产品)用于第一批50L反应器培养制备高ADCC活性抗体
复苏：从液氮罐中抽取细胞(冻存管内保存)一支(装量1.5mL)，37℃水浴化冻，立即将种子液转入含有20～30mL CD FortiCHO培养基的125mL摇瓶中。将摇瓶放置于CO2培养箱中，37℃，8％CO2，湿度80％，130rpm培养3～4天。
摇瓶扩种：当细胞密度增加至2×106～4×106cells/mL时，对细胞进行扩种。操作时根据需要，补充适量的新鲜CD Dynamis培养基，扩种后细胞的密度在0.4×106～0.6×106cells/mL。根据细胞液体积更换适宜体积的摇瓶，培养体积不超过摇瓶总体积的1/3。
15L反应器N-1代种子制备：反应器为Applikon EZ Control 15L玻璃罐，参数控制：(1)pH控制：7.0±0.1，分别用CO2和0.5M NaHCO3PID反馈控制；(2)DO控制：设定40±10％，用纯氧气PID反馈控制；(3)转速控制：80～200rpm，起始转速80rpm，接种后1hr，转速提高至120rpm。(4)温度控制：36.5℃±0.5℃；(5)气体控制：Overlay压缩空气，恒速50～100mL/min；Sparger CO2PID反馈控制，CO2设定上限300mL/min，Sparger O2PID反馈控制，O2设定上限300mL/min。接种5.5～6.5L，接种后密度控制在0.5×106cells/mL左右，培养4～5天，密度至4～6×106cells/mL时，进入到50L SUB或者50L Mobius反应器中试放大培养。
50L SUB反应器操作：接种前先准备好Applikon控制器和50L SUB袋及配套管路(均为无菌)，配制好20L CD Dynamis培养基经过滤打入袋中，培养基含2-氟-过乙酰基-岩藻糖(2F-Peracetyl-Fucose)50～200μM，温度控制为36.5±0.5℃，表面通气50～100mL/min，深层Sparger空气为100mL/min，转速80rpm，过夜校正溶氧为100％。校正溶氧2小时后，DO≥95％，接种密度为4～6×106cells/mL的种子液25L，接种后密度为0.8～1.2×106cells/mL，深层Sparger微泡通氧气、深层Sparger大泡通空气和CO2，深层Sparger大泡空气通过监控代谢CO2浓度进行调节，大泡通气量范围为0～1500mL/min，开启PID控制，pH通过CO2和外接NaHCO3调节，培养过程中根据泡沫情况添加消泡剂50～100ppm。培 养参数设定：DO 40±10％，pH 7.0±0.1，温度36.5±0.5℃，接种30min后，取样检测细胞密度和活率，转速80～100rpm。培养过程中流加EFC、葡萄糖。培养周期14～16天，活率约70％结束培养，收获发酵液。发酵液纯化主要步骤包括深层过滤、亲和层析、酸孵育灭活、阳离子交换层析、阴离子交换层析、纳滤及超滤步骤。纯化后的样品送检糖型分析。
2-氟-过乙酰基-岩藻糖(2F-Peracetyl-Fucose)于50L SUB反应器第一批中试根据表6设计的补料策略和培养条件进行添加和控制：
表6
Figure PCTCN2016089820-appb-000007
备注：1、初始L-谷氨酰胺添加浓度6mM、葡萄糖控制2～4g/L；
2、细胞接种后密度控制在0.8～1.2×106cells/mL；
3、EFC：第3天开始每天按初始工作体积在线补加3％(V/V)，补加总量为33～36％；
4、2-脱氧-2-氟-L-岩藻糖于第0天添加至基础培养基中，浓度为100μM；
5、Tyr于第0天添加至基础培养基中，添加量为1/500(工作体积)，Tyr配制浓度为100g/L。
实施例7：5-炔基-L-岩藻糖(5-Alkynyl-Fucose，CAS号：1193251-61-4)于第二批50L反应器培养制备高ADCC活性抗体
复苏：从液氮罐中抽取细胞(冻存管内保存)一支(装量1.5mL)，37℃水浴化冻，立即将种子液转入含有20～30mL CD FortiCHO培养基的125mL摇瓶中。将摇瓶放置于CO2培养箱中，37℃，8％CO2，湿度80％，130rpm培养3～4天。
摇瓶扩种：当细胞密度增加至2×106～4×106cells/mL时，对细胞进行扩种。操作时根据需要，补充适量的新鲜CD FortiCHO培养基，扩种后细胞的密度在0.4×106～0.6×106cells/mL。根据细胞液体积更换适宜体积的摇瓶，培养体积不超过摇瓶总体积的1/3。
15L反应器N-1代种子制备：反应器为Applikon EZ Control 15L玻璃罐，参数控制：(1)pH控制：7.0±0.1，分别用CO2和0.5M NaHCO3PID反馈控制；(2)DO控制：设定40±10％，用纯氧气PID反馈控制；(3)转速控制：80～200rpm，起始转速80rpm，接种后1hr，转速提高至120rpm。(4)温度控制：36.5℃±0.5℃；(5)气体控制：Overlay压缩空气，恒速50～100mL/min；Sparger CO2PID反馈控制，CO2设定上限300mL/min， Sparger O2PID反馈控制，O2设定上限300mL/min。接种5.5～6.5L，接种后密度控制在0.5×106cells/mL左右，培养4～5天，密度至4×106～6×106cells/mL时，进入到50L SUB或者50L Mobius反应器中试放大培养。
50L Mobius反应器操作：接种前先准备好Applikon控制器和50L SUB袋及配套管路(均为无菌)，配制好20L CD FortiCHO培养基经过滤打入袋中，培养基含5-炔基-L-岩藻糖(5-Alkynyl-Fucose)50～200μM，温度控制为36.5±0.5℃，表面通气50-100mL/min，深层Sparger空气为100mL/min，转速80rpm，过夜校正溶氧为100％。校正溶氧2小时后，DO≥95％，接种密度为4×106～6×106cells/mL的种子液25L，接种后密度为0.8×106～1.2×106cells/mL，深层Sparger微泡通氧气、深层Sparger大泡通空气和CO2，深层Sparger大泡空气通过监控代谢CO2浓度进行调节，大泡通气量范围为0～1500mL/min，开启PID控制，pH通过CO2和外接NaHCO3调节，培养过程中根据泡沫情况添加消泡剂50～100ppm。培养参数设定：DO 40±10％，pH 7.0±0.1，温度36.5±0.5℃，接种30min后，取样检测细胞密度和活率，转速80～100rpm。培养过程中流加EFC、Gluc。培养周期14～16天，活率约70％结束培养，收获发酵液。发酵液纯化主要步骤包括深层过滤、亲和层析、酸孵育灭活、阳离子交换层析、阴离子交换层析、纳滤及超滤步骤。纯化后的样品送检糖型分析。
5-炔基-L-岩藻糖于50L Mobius反应器第二批中试培养根据表7设计的补料策略和培养条件进行添加和控制：
表7
Figure PCTCN2016089820-appb-000008
备注：1、初始L-谷氨酰胺添加浓度6mM、葡萄糖控制2～4g/L；
2、细胞接种后密度控制在0.8×106～1.2×106cells/mL；
3、EFC：第3天开始每天按初始工作体积在线补加3％(V/V)，补加总量为33～36％；
4、5-炔基-L-岩藻糖于第0天添加至基础培养基中，浓度为100μM；
5、Tyr于第0天添加至基础培养基中，添加量为1/500(工作体积)，Tyr配制浓度为100g/L。
实施例8：2-脱氧-2-氟-L-岩藻糖(2-Deoxy-2-fluoro-L-Fucose，CAS号：70763-62-1)于第三批50L反应器培养制备高ADCC活性抗体
复苏：从液氮罐中抽取细胞(冻存管内保存)一支(装量1.5mL)，37℃水浴化冻， 立即将种子液转入含有20～30mL CD FortiCHO培养基的125mL摇瓶中。将摇瓶放置于CO2培养箱中，37℃，8％CO2，湿度80％，130rpm培养3～4天。
摇瓶扩种：当细胞密度增加至2×106～4cells/mL时，对细胞进行扩种。操作时根据需要，补充适量的新鲜CD FortiCHO培养基，扩种后细胞的密度在0.4×106～0.6cells/mL。根据细胞液体积更换适宜体积的摇瓶，培养体积不超过摇瓶总体积的1/3。
15L反应器N-1代种子制备：反应器为Applikon EZ Control 15L玻璃罐，参数控制：(1)pH控制：7.0±0.1，分别用CO2和0.5M NaHCO3PID反馈控制；(2)DO控制：设定40±10％，用纯氧气PID反馈控制；(3)转速控制：80～200rpm，起始转速80rpm，接种后1hr，转速提高至120rpm。(4)温度控制：36.5℃±0.5℃；(5)气体控制：Overlay压缩空气，恒速50～100mL/min；Sparger CO2PID反馈控制，CO2设定上限300mL/min，Sparger O2PID反馈控制，O2设定上限300mL/min。接种5.5～6.5L，接种后密度控制在0.5×106cells/mL左右，培养4～5天，密度至4×106～6×106cells/mL时，进入到50L SUB或者50L Mobius反应器中试放大培养。
50L Mobius反应器操作：接种前先准备好Applikon控制器和50L SUB袋及配套管路(均为无菌)，配制好20L CD Dynamis培养基经过滤打入袋中，培养基含2-脱氧-2-氟-L-岩藻糖(2-Deoxy-2-fluoro-L-Fucose)50～200μM，温度控制为36.5±0.5℃，表面通气50～100mL/min，深层Sparger空气为100mL/min，转速80rpm，过夜校正溶氧为100％。校正溶氧2小时后，DO≥95％，接种密度为4×106～6×106cells/mL的种子液25L，接种后密度为0.8×106～1.2×106cells/mL，深层Sparger微泡通氧气、深层Sparger大泡通空气和CO2，深层Sparger大泡空气通过监控代谢CO2浓度进行调节，大泡通气量范围为0～1500mL/min，开启PID控制，pH通过CO2和外接NaHCO3调节，培养过程中根据泡沫情况添加消泡剂50～100ppm。培养参数设定：DO 40±10％，pH 7.0±0.1，温度36.5±0.5℃，接种30min后，取样检测细胞密度和活率，转速80～100rpm。培养过程中流加EFC、Gluc。培养周期14～16天，活率约70％结束培养，收获发酵液。发酵液纯化主要步骤包括深层过滤、亲和层析、酸孵育灭活、阳离子交换层析、阴离子交换层析、纳滤及超滤步骤。纯化后的样品送检糖型分析、ADCC活性检测等。
2-脱氧-2-氟-L-岩藻糖(2-Deoxy-2-fluoro-L-Fucose)于50L Mobius反应器第三批中试根据表8设计的补料策略和培养条件进行添加和控制：
表8
Figure PCTCN2016089820-appb-000009
备注：1、初始L-谷氨酰胺添加浓度6mM、葡萄糖控制2～4g/L；
2、细胞接种后密度控制在0.8-1.2×106cells/mL；
3、EFC：第3天开始每天按初始工作体积在线补加3％(V/V)，补加总量为33-36％；
4、2-脱氧-2-氟-L-岩藻糖于第0天添加至基础培养基中，浓度为100μM；
5、Tyr于第0天添加至基础培养基中，添加量为1/500(工作体积)，Tyr配制浓度为100g/L。
实施例9：糖苷分析
分别取500μg实施例6～8中所获得的样品蛋白加入1×PBS至500μL(1μg/μL)混匀；加入PNGaseF酶5μL，混匀后37℃水浴反应3h。
Waters小柱(Glyco Works 1cc P.N.186007080)中加入1000μL超纯水，抽出；加入1000μL 85％乙腈，抽出进行预处理；向经PNGaseF酶处理的去糖基化样品中加入3500μL乙腈，混匀后(混匀时可能出现浑浊，勿离心)注入该柱中；用400μL 85％ACN淋洗小柱4次，流穿液弃去；用100μL 5％ACN/NH4HCO3洗脱多聚糖，重复3次，收集流穿液，获得糖苷；将接取的目标液冻干；取200μL 1％甲酸复溶冻干样品；室温孵育40min；冻干；取300μL醋酸与700μL DMSO混匀；取出800μL加入1mg 2-AB(本领域公知的荧光物质)混匀；加入12mg氰基硼氢化钠混匀(需在1小时内新鲜配制)；取300μL复溶冻干样品，65℃孵育3小时；孵育后样品加入3000μL ACN混匀，其余过柱步骤相同，冻干，获得2-AB标记的糖苷样品。冻干后样品，分析前用160μL 60％乙腈复溶，13000rpm离心2min，取150μL上清分析。
糖苷标准品Glycan Test(waters，P.N.186006349，S.N.W19011504，250μL 60％乙腈复溶)及2-AB标记样品进行具荧光检测器的高效液相色谱分析，色谱条件如下表9所示：
表9
Figure PCTCN2016089820-appb-000010
代表性糖苷(实施例8所得抗体)色谱如图7所示：
实施例6～8三批糖苷分析结果见下表10所示：
表10
Figure PCTCN2016089820-appb-000011

Claims (20)

  1. 一种通过改变奥法木(ofatumumab)单抗的糖链组成提升其生物学活性的修饰抗体。
  2. 根据权利要求1所述的修饰抗体，其中，所述修饰抗体的一级氨基酸序列与奥法木(ofatumumab)单抗一级氨基酸序列一致，但其在表达过程中修饰抗体的糖链与奥法木单抗的糖链不同，使其生物学活性较奥法木抗体显著增强，例如提升10～100倍。
  3. 根据权利要求2所述的修饰抗体，其中，通过降低修饰抗体的糖链中岩藻糖含量，增强抗体依赖性细胞介导的细胞毒性。
  4. 根据权利要求3所述的修饰抗体，其中，在由碳水化合物所组成的修饰抗体的糖链中，具岩藻糖的糖链占修饰抗体的总糖链的质量百分比不高于20％。
  5. 根据权利要求3所述的修饰抗体，其中，在由碳水化合物所组成的修饰抗体的糖链中，具岩藻糖的糖链占修饰抗体的总糖链的质量百分比不高于10％，例如其质量百分比不高于8％。
  6. 根据权利要求4所述的修饰抗体，其中，在由碳水化合物所组成的修饰抗体的糖链中，具岩藻糖的糖链占修饰抗体的总糖链的质量百分比不高于5％，例如0.2％～5％。
  7. 根据权利要求6所述的修饰抗体，其中，由碳水化合物所组成的修饰抗体的糖链中，不含岩藻糖。
  8. 权利要求1～7中任一项所述的修饰抗体的制备方法，其中，所述方法包括由能表达奥法木单抗的真核宿主细胞在添加了L-岩藻糖类似物的培养基中翻译表达的步骤。
  9. 根据权利要求8所述的方法，其中，所述L-岩藻糖类似物包括5-炔基-L-岩藻糖、2-脱氧-2-氟-L-岩藻糖、2-氟-过乙酰基-岩藻糖(2F-Peracetyl-Fucose)和它们相应的过酰化衍生物及其他的衍生物中的一种或多种。
  10. 根据权利要求9所述的方法，其中，所述L-岩藻糖类似物包括2-脱氧-2-氟-L-岩藻糖和/或相应的过酰化衍生物及其他衍生物。
  11. 根据权利要求10所述的方法，其中，所述岩藻糖类似物包括5-炔基-L-岩藻糖和/或相应的过酰化衍生物或其他衍生物。
  12. 根据权利要求8所述的方法，其中，所述能表达奥法木单抗的真核宿主细胞包括CHO细胞系、NS0细胞系或SP2/0细胞系。
  13. 根据权利要求12所述的方法，其中，所述能表达奥法木单抗的真核宿主细胞为CHO细胞系。
  14. 一种药物组合物，所述组合物包含权利要求1～7中任一项所述的修饰抗体。
  15. 权利要求1～7中任一项所述的修饰抗体或权利要求14中所述的药物组合物在制备抗肿瘤药物中的应用。
  16. 根据权利要求15所述的应用，其中，所述的肿瘤选自慢性淋巴瘤、非霍奇金氏淋巴瘤和/或弥漫性大B细胞淋巴瘤。
  17. 权利要求8～13中任一项所述的方法在制备抗肿瘤药物中的应用。
  18. 根据权利要求17所述的应用，其中，所述的肿瘤选自慢性淋巴瘤、非霍奇金氏淋巴瘤和/或弥漫性大B细胞淋巴瘤。
  19. 一种治疗肿瘤的方法，所述方法包括给予受试体有效剂量的权利要求1～7中任一项所述的修饰抗体或权利要求14所述的药物组合物。
  20. 根据权利要求19所述的方法，其中，所述肿瘤选自慢性淋巴瘤、非霍奇金氏淋巴瘤和/或弥漫性大B细胞淋巴瘤。
PCT/CN2016/089820 2015-07-13 2016-07-12 抗体依赖性细胞介导的细胞毒性增强的奥法木抗体 WO2017008736A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201680028044.2A CN108473570A (zh) 2015-07-13 2016-07-12 抗体依赖性细胞介导的细胞毒性增强的奥法木抗体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510406202.0 2015-07-13
CN201510406202.0A CN106699886A (zh) 2015-07-13 2015-07-13 抗体依赖性细胞介导的细胞毒性(adcc)增强的奥法木抗体

Publications (1)

Publication Number Publication Date
WO2017008736A1 true WO2017008736A1 (zh) 2017-01-19

Family

ID=57756716

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/089820 WO2017008736A1 (zh) 2015-07-13 2016-07-12 抗体依赖性细胞介导的细胞毒性增强的奥法木抗体

Country Status (2)

Country Link
CN (2) CN106699886A (zh)
WO (1) WO2017008736A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112899235A (zh) * 2019-12-03 2021-06-04 信达生物制药(苏州)有限公司 一种细胞培养液的收获方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2022003030A (es) * 2019-09-11 2022-04-07 Novartis Ag Tratamiento de afecciones distintas de la esclerosis multiple en pacientes tratados con ofatumumab.

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101945667A (zh) * 2007-12-21 2011-01-12 健泰科生物技术公司 利妥昔单抗不应性类风湿性关节炎患者的疗法
CN102076865A (zh) * 2008-05-02 2011-05-25 西雅图基因公司 用于制造核心岩藻糖基化降低的抗体和抗体衍生物的方法和组合物
CN103402525A (zh) * 2010-08-05 2013-11-20 西雅图基因公司 使用岩藻糖类似物在体内抑制蛋白质岩藻糖基化的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101945667A (zh) * 2007-12-21 2011-01-12 健泰科生物技术公司 利妥昔单抗不应性类风湿性关节炎患者的疗法
CN102076865A (zh) * 2008-05-02 2011-05-25 西雅图基因公司 用于制造核心岩藻糖基化降低的抗体和抗体衍生物的方法和组合物
CN103402525A (zh) * 2010-08-05 2013-11-20 西雅图基因公司 使用岩藻糖类似物在体内抑制蛋白质岩藻糖基化的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112899235A (zh) * 2019-12-03 2021-06-04 信达生物制药(苏州)有限公司 一种细胞培养液的收获方法

Also Published As

Publication number Publication date
CN106699886A (zh) 2017-05-24
CN108473570A (zh) 2018-08-31

Similar Documents

Publication Publication Date Title
CN102216452B (zh) 具有变异糖基化方式的细胞系和蛋白质
AU2017206006B2 (en) Modulation of afucosylated species in a monoclonal antibody composition
WO2005053742A1 (ja) 抗体組成物を含有する医薬
CN1071836A (zh) 治疗肿瘤病的药品及其制备方法
EP3590534A1 (en) Method for treating egfr-tki-resistant non-small cell lung cancer by administration of anti-her3 antibody-drug conjugate
CN114478806B (zh) 一种提升免疫细胞杀伤活性的嵌合受体及其应用
CN111534475B (zh) 一种细胞表面偶联抗体的方法及其应用
ES2893536T3 (es) Método para preparar anticuerpo mediante la regulación del contenido de azúcares del anticuerpo
US20190048070A1 (en) Reduction of high molecular weight species, acidic charge species and fragments in a monoclonal antibody composition
WO2020147297A1 (zh) 一种抗egfr全人源化单克隆抗体的纯化工艺
WO2017008736A1 (zh) 抗体依赖性细胞介导的细胞毒性增强的奥法木抗体
WO2020147298A1 (zh) 一种用于制备抗egfr全人源化单克隆抗体的细胞株构建方法
CN107460221B (zh) 一种降低抗pd-l1抗体中蛋白聚合物的细胞培养方法
Liu et al. Combined effect of lentinan and cisplatin on cytokines IL-6, TNF-α, and TGF-β in Tumor Therapy
CN114369060B (zh) 吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用
US20230045902A1 (en) Composition for reinforcing function of stem cell
CN114621327B (zh) GLP-1、GIP和Gcg多重受体激动蛋白质
CN106119192B (zh) 组合物及其在cik细胞培养中的应用
WO2015020567A1 (en) Laminaria angustata-based product for enhancing regeneration and proliferation of cells, method of producing and use the same
KR101980954B1 (ko) 포르피로모나스 긴기발리스의 53-kDa 단백질에 대한 항체 생산용 조성물 및 이의 용도
CN116615231A (zh) Fab高甘露糖糖型
CN115843784A (zh) 一种car-nk细胞保存液及其制备方法、保存方法
CN112011514A (zh) 提高抗体adcc活性的细胞培养工艺
CN112439079A (zh) 有机溶剂法制备载药笼状蛋白
CN117122606A (zh) 淫羊藿次苷i在治疗或预防微卫星稳定型实体瘤中的应用及包含其的药盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16823883

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16823883

Country of ref document: EP

Kind code of ref document: A1