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WO2015101163A1 - Application de billes immunomagnétiques se couplant à un anticorps monoclonal anti-hla-g en tri de cellules tumorales - Google Patents

Application de billes immunomagnétiques se couplant à un anticorps monoclonal anti-hla-g en tri de cellules tumorales Download PDF

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WO2015101163A1
WO2015101163A1 PCT/CN2014/093816 CN2014093816W WO2015101163A1 WO 2015101163 A1 WO2015101163 A1 WO 2015101163A1 CN 2014093816 W CN2014093816 W CN 2014093816W WO 2015101163 A1 WO2015101163 A1 WO 2015101163A1
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hla
beads
tumor cells
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卢英
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells

Definitions

  • the invention relates to a method for sorting tumor cells, and a method for sorting tumor cells by using magnetic beads coated with anti-human leukocyte antigen-G (HLA-G) antibody.
  • HLA-G anti-human leukocyte antigen-G
  • the number of circulating tumor cells in the peripheral blood is extremely small, usually only a few tumor cells in about 100 million white blood cells and 50 billion red blood cells, and only one circulating tumor cells per 10 5 to 10 7 monocytes. Therefore, in order to increase the detection rate of circulating tumor cells, it is usually necessary to perform enrichment of circulating tumor cells before detection.
  • Cell enrichment can be achieved by specific markers (immunoisolation) of tumor cells or cell morphological features such as cell volume and density.
  • Commonly used cell enrichment techniques include immunomagnetic sorting, density gradient centrifugation, and cell filtration.
  • Density Gradient Centrifugation It is a technique based on physical properties to separate cells by different sedimentation rates between cells under a certain centrifugal force. This is a commonly used technique in the laboratory for the separation of monocytes and the like.
  • the Onclquick (Greiner Bio-One, Frickenhausen, Germany) and Ficoll-Hypaque separation methods established by this technique were used to separate tumor cells under a certain centrifugal force using a dedicated porous barrier and density gradient separation solution.
  • the device is simple and easy to operate, and can obtain the complete morphology of the cells, which is convenient for further identification. It shows certain clinical significance in some clinical studies, but the sensitivity and specificity are not high, and the recovery rate of the fat tumor cells is low, which limits Its clinical application.
  • Membrane filtration method the difference between the diameter of the tumor cells and the blood cells is utilized, and the cells are filtered through a membrane having a certain pore size to achieve the purpose of separating the cells.
  • this technique is a technique that uses the morphological features of cells to enrich tumor cells.
  • the blood cell diameter is between 8-llum, and the tumor cells are relatively large in diameter (for example, breast cancer cells are about 30 um in diameter), thereby distinguishing between tumor cells and normal blood cells.
  • the difference between tumor cells and some blood cells is not so obvious. It is the aspect of the technology being humanized, but its advantages are equally obvious: more types of tumors are suitable for using size to distinguish normal blood cells without missing surface markers.
  • the first method to use this method is ISET (Isolation by Size of Epithilial Tumor cells), which is simple in equipment, easy to operate, and the cell surface markers can be completely retained; the aggregated tumor cells are more easily separated, which is beneficial for further follow-up. Detection.
  • ISET Isolation by Size of Epithilial Tumor cells
  • the pore size of the tumor cells in the circulation is not uniform, and deformation is often caused by the attack of the immune system and the shearing force of the blood, so the size setting of the membrane pore size is a problem.
  • the microfiltration technology for separating tumor cells according to the cell size has been successfully developed, and the initial experimental results show that it is more sensitive than the antigen-antibody binding separation method, and is more convenient for molecular detection.
  • the following is a specific description.
  • Cell size-based microchip filtration The enrichment platform based on tumor cell size has been reported more and more, and the tumor cells obtained by this method are beneficial for the next step of gene and molecular analysis.
  • One of the high-density micropore techniques greatly improved the enrichment rate of circulating tumor cells in this method.
  • the cultured cells were added to peripheral blood and found to recover more than 90% of tumor cells, and 7.5 mL of blood samples. Separation takes less than 3 minutes to complete.
  • the chip found tumor cells in 51 patients, while the CellSearch system was only positive in blood samples from 26 patients. And the number of circulating tumor cells recovered by the chip is 5.5 times that of the CellSearch system.
  • Antigen-antibody binding separation method Circulating tumor cell separation method based on antigen-antibody binding is currently the most commonly used method. Using different surface antigen markers expressed by tumor cells and normal blood cells, different antibodies are designed to bind to them, and the high specificity and sensitivity of antigen-antibody binding can be used to distinguish and enrich circulating tumor cells (mostly antibodies and The magnetic beads combine to achieve the purpose of separation in a magnetic field. The effectiveness of the immunomagnetic bead enrichment method is much higher than the traditional method. Compared with many enrichment techniques, this technology is relatively mature and has high cell recovery rate. The reported tumor cell recovery rate is about 85%.
  • the magnetic bead-based enrichment method is divided into two principles: immunopositive enrichment and negative enrichment.
  • EpCAM epithelial cell adhesion factor
  • EpCAM epithelial cell adhesion factor
  • EMT epithelial-mesenchymal transition
  • the newly developed biochip technology using EpCAP-coated chip microgrooves, can filter blood-enriched tumor cells and increase enrichment efficiency by about 100 times.
  • EMT epithelial to mesenchymal transition
  • EpCAM epithelial marker antigen
  • one of the characteristics of tumor cells is heterogeneity.
  • Some CTCs do not express EpCAM, and positive selection may lead to false negative results.
  • the quality and quantity of the magnetic beads will affect their identification.
  • the volume of the magnetic beads has been reduced by several tens of times, and this effect is not obvious.
  • Immunomagnetic beads negative sorting similar to positive capture, negative sorting also uses the principle of magnetic bead sorting. However, antibodies that bind negatively to magnetic beads are antigens that bind to the surface of normal blood cells, and tumor cells do not express such antigens on the surface, such as CD45. The principle of negative enrichment is to remove all other cells in the blood, and then the remaining cells are all rare cells including CTCs, and then identified. The red blood cells in the blood are lysed, and the white blood cells bind to the antibody and the magnetic beads and remain in the magnetic field. The rest are tumor cells and a small amount of red and white blood cells, and then the tumor cells are identified and counted by immunostaining.
  • the disadvantage of negative enrichment is that the recovery rate is less than positive enrichment.
  • the advantage is that the identified CTCs are not labeled by magnetic beads and are relatively less affected during sample processing and enrichment. Compared with positive capture, negative sorting is not affected by changes in tumor cell surface antigens, so it can also be applied to tumors with inconsistent and specific specificity and sensitivity of circulating tumor cells, such as melanoma. The sensitivity is relatively high, but it interferes with more cell residues. This technology has not developed an automatic system at present, and the operation is relatively complicated. The influence of human factors is relatively large, but it is still a possible future development direction.
  • the CellSearch system is currently the only FDA-approved circulating tumor cell enrichment technology that has been approved for large-scale clinical validation for prognostic evaluation and efficacy testing of metastatic adenocarcinoma, colorectal cancer, and prostate cancer. A number of studies have confirmed that the system can effectively predict the survival of patients with metastatic breast, colorectal and prostate cancer, and more importantly, can predict the efficacy early after routine treatment in a cycle, so it has been approved by the FDA for the above several advanced stages. Tumor patients.
  • the CellSearch system is an automated circulatory spleen cell detection system whose detection stability has been validated in multiple centers. The system uses the principle of immunomagnetic beads positive capture.
  • the workflow is: using immunomagnetic beads combined with EpCAM antibody to bind tumor cells in a magnetic field, and then using keratin (CK) antibody to detect circulating tumor cells, and also can determine CD45, with CrCD45_ as the tumor cell standard Quasi, finally count the circulating tumor cells.
  • the system can also give partial cell morphology data such as nuclear cytoplasmic ratio, and can collect the obtained tumor cells for artificial identification.
  • the CellSearch system typically requires 7.5 ml of peripheral blood to perform the above operations, and can additionally detect HER2 expression if needed, but with a general ability.
  • Immunomagnetic beads have been widely used in the detection of circulating tumor cells due to their high sensitivity, specificity and rapid separation.
  • EMT epithelial-mesenchymal transition
  • some epithelial-specific antigen expression is weakened when tumor cells enter the circulatory system. Or disappear; while the tumor cells entering the circulatory system under the influence of human autoimmunity and hemorheology, their own apoptosis and decreased antigen expression may lead to a weakening of the ability of positive capture technology to detect circulating tumor cells. Therefore, this technique has certain limitations for certain tumors.
  • Antigen-antibody-binding microstrip technology Several microchip technologies have been reported for spleen tumor cell detection. Recently, the microchips have been studied by surface-arranged coated EpCAM antibodies. The recycling efficiency is over 60%. The chip can be applied to the detection of a variety of tumor circulating tumor cells, including lung cancer, prostate cancer, pancreatic cancer, breast cancer and colorectal cancer, and has been verified in the prediction experiment of lung cancer. The tumor cells obtained by this technology can be used. The next step in the detection of EGFR. The chip technology requires blood flow at a very slow rate to allow antigenic antibodies to bind well, and 7.5 ml of blood takes approximately 10 h.
  • New technologies based on microfluidic technology and cell size separation are also being developed.
  • blood samples are passed through a certain space of the pores, and then arranged through the microcolumns to form a space of a certain size and spacing.
  • Cells above a certain size are retained, and others are retained. Is separated. Separation of different cells and plasma can be achieved using pores of different spacing.
  • microelectrode array technology has facilitated the development of two-dimensional electrophoretic separation technology to enrich circulating tumor cells.
  • Cells with different characteristics will find their own position at different distances of the microelectrodes and accumulate cells with different characteristics because the difference between hydrodynamics and conductivity is separated, and the characteristics of the cells themselves are completely protected.
  • the cultured tumor cells were used to test the enrichment rate of the technique, and the recovery rate was found to exceed 90%.
  • the shortcoming of these techniques is that the amount of blood used is too small, and must be diluted with the corresponding isotonic working solution. If the experiment is carried out according to the blood volume commonly used in clinical practice, the working fluid amount can reach 70 ml, and the passing speed is only 0.5. Ml / h, the dilution process of the liquid will also cause the loss of circulating tumor cells.
  • HLA-G leukocyte antigen G
  • the immune system has the function of repelling and eliminating these cancerous cells, which is called host immune surveillance.
  • cancer cells have clearly escaped the host's immune surveillance. This phenomenon is called the immune escape of tumors, and the mechanism is that tumor cells have an anti-ancestor phenomenon in the early stage, which can secrete and express. HLA-G, which allows it to escape the host's immune surveillance and killing.
  • HLA-G is abnormally expressed only in malignant tumors, and is not expressed in normal tissues other than the placenta. It is a new tumor marker with high malignant tumor specificity.
  • the conference summarized the progress of the HLA-G study since the first academic seminar in 1999, clearly stating that the abnormal expression of HLA-G in tumor cells is Indisputable facts.
  • the technical problem to be solved by the present invention provides a method for sorting breast cancer tumor cells.
  • the tumor cells include breast cancer tumor cells, esophageal cancer, non-small cell lung cancer, uterine cancer, gastric cancer, colon cancer and the like.
  • a method for sorting tumor cells comprising the following steps:
  • the anti-HLA-G monoclonal antibody to which the magnetic beads are coupled is MEM-G/2, MEM-G/9 or 4H84.
  • the anti-HLA-G monoclonal antibody and the immunomagnetic beads are subjected to antibody saturation coupling, preferably using a mass ratio of 200 ug antibody: 1000 ug to activate the magnetic beads.
  • the preparation of the immunomagnetic beads taking activated magnetic beads, adding a saturated coupling amount of anti-HLA-G monoclonal antibody and PBS, mixing at room temperature for 3 hours, washing three times with PBS, adding glycine to mix, The remaining aldehyde groups were blocked; mixed with BSA-containing PBS solution for 30 min, the non-specific adsorption sites were blocked, washed three times with PBS, and vortexed with PBS buffer to obtain a uniform modified immunomagnetic beads.
  • the volume ratio of the cell fluid to be sorted to the HLA-G immunomagnetic beads is 100: (20-100).
  • the present invention also provides an anti-HLA-G immunomagnetic bead for isolating tumor cells. And a sorting kit containing anti-HLA-G immunomagnetic beads.
  • HLA-G was used to sort tumor cell markers.
  • HLA-G was a tumor-specific broad-spectrum marker. After coupling with immunomagnetic beads, the specificity was good and the tumor efficiency was high.
  • HLA-G coupled with immunomagnetic beads has good sensitivity and reproducibility, and can detect relevant tumor cells.
  • HLA-G monoclonal antibodies MEM-G/2, MEM-G/9 and 4H84, source company: Santa Cruz Biotechnology.
  • Nanoimmune magnetic bead preparation involves the coupling of activated magnetic beads to the monoclonal antibody. By optimizing the particle size of the nanoparticles and the attachment efficiency of the antibody, the adsorption efficiency of the nano-immunized magnetic beads on the antigen cells is improved.
  • the magnetic beads are selected to activate the magnetic beads at 50 nm.
  • Antibody coupling 1) Before antibody coupling, anti-HLA-G antibody (using MEM-G/2, MEM-G/9 and 4H84, respectively) to PBS at 4 ° C, after three times of dialysis, overnight, Store in PBS and adjust the concentration to 5mg/ml.
  • reaction medium 500 ⁇ L of nano magnetic beads were placed in 7 centrifuge tubes, numbered, and the medium in tubes 1 and 2 was replaced with phosphate buffer solution under the action of external magnetic field.
  • 0.1M PB 50 ⁇ L of nano magnetic beads were placed in 7 centrifuge tubes, numbered, and the medium in tubes 1 and 2 was replaced with phosphate buffer solution under the action of external magnetic field.
  • 0.1M PB 50 ⁇ L of nano magnetic beads were placed in 7 centrifuge tubes, numbered, and the medium in tubes 1 and 2 was replaced with phosphate buffer solution under the action of external magnetic field.
  • 40 ⁇ L (5mg/ml) antibody was added
  • the content of the antibody in the supernatant after the coupling reaction was detected by using a (BCA) kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.) in combination with a microplate reader (PerKin EImer, USA).
  • the anti-HLA-G monoclonal antibody solution was diluted to a standard solution of 0.5 mg/ml with a pH of 7.4, 0.01 mol of phosphate tween (0.05% Tween-20) solution, and taken as 0, 1, 2, 4, 8, respectively.
  • a standard curve is prepared according to the concentration of the standard solution and the absorbance value, and the amount of the antibody in the supernatant is obtained according to the absorbance value of the supernatant to be detected, thereby obtaining the antibody content coupled to the surface of the magnetic bead per unit mass.
  • reaction medium The suitable ion concentration and pH of the reaction medium play an important role in the efficient coupling of magnetic beads with proteins to reduce non-specific adsorption between them.
  • the experimental results show that all three antibodies are suitable.
  • Optimal coating time It can be seen from Table 1 that the three antibodies basically reached saturation after being adsorbed by the magnetic beads for 3 hours, and the OD280 value tends to be stable, so the coating time was determined to be 3 hours.
  • Table 1 The best time for antibody coated magnetic beads (take 4H84 as an example)
  • antibody magnetic bead coupling ratio the results finally showed anti-HLA-G 4H84 monoclonal antibody, the antibody concentration was set to 5mg / ml, the magnetic bead concentration was 2mg / ml, the antibody and magnetic beads mass ratio was 200ug: 1000ug, Magnetic bead coupling is performed at this time, and antibody coupling can be substantially saturated.
  • Magnetic Bead Surface Antibody Quantification The amount of anti-HLA-G antibody attached to the surface of the magnetic beads was measured using a BCA protein quantification kit. The absorbance value corresponding to the protein standard solution having a known concentration is obtained. According to the amount of protein contained in different standard solutions and their absorbance values, a standard curve is made.
  • the absorbance value of the supernatant sample is substituted into the formula, and the protein content in the supernatant sample is 71.2, respectively, because the reaction antibody is 200 ⁇ g, that is, the amount of antibody attached per 1000 ug of magnetic beads is 128.8; It was calculated that the amount of anti-HLA-G antibody attached to the surface of the magnetic beads was 128.8 ⁇ g/mg on average.
  • the three anti-HLA-G immunomagnetic beads prepared in step 2.2 of the present embodiment were used for artificially cultured tumor cell line cells for tumor cell collection; the artificially cultured tumor cell line cells were mixed with healthy human blood to prepare artificial samples, and used. Anti-HLA-G immunomagnetic beads were used to enrich tumor cells in artificial samples, and the enriched samples were counted and the enrichment efficiency was calculated.
  • the anti-HLA-G immunomagnetic beads can achieve a recovery rate of 85%-93% for the corresponding tumor cell enrichment rate.
  • the enrichment rate of tumor cells in blood can reach 77%-84%, which indicates that the prepared anti-HLA-G immunomagnetic beads have higher enrichment rate and better reproducibility, and can recover related tumor cells.
  • the amount of nano-immunized magnetic beads see Table 3 tested, the ratio of tumor sample to nano-magnetic beads is 100ul (10 7 /ml): 40ul (2mg / ml), the enrichment effect is better and will not cause magnetic bead waste .
  • the optimal reaction time of nano-immunized magnetic beads and tumor cells see Table 4.
  • the optimal time for the reaction of the nano-immunized magnetic beads with the sample is 30 min.

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Abstract

L'invention porte sur une application de billes immunomagnétiques se couplant à un anticorps monoclonal anti-HLA-G en tri de cellules tumorales. L'invention porte également sur un procédé pour le tri de cellules tumorales. Le procédé comprend les étapes suivantes : (1) l'obtention de billes immunomagnétiques HLA-G à partir de billes immunomagnétiques se couplant à un anticorps monoclonal anti-HLA-G ; (2) le mélange homogène des billes immunomagnétiques HLA-G, puis l'ajout de celles-ci à du suc cellulaire à trier et la séparation de cellules tumorales à l'aide d'un procédé de séparation de billes immunomagnétiques.
PCT/CN2014/093816 2013-12-31 2014-12-15 Application de billes immunomagnétiques se couplant à un anticorps monoclonal anti-hla-g en tri de cellules tumorales Ceased WO2015101163A1 (fr)

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CN201310749974.5A CN103756967B (zh) 2013-12-31 2013-12-31 抗hla-g的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用
CN201310749974.5 2013-12-31

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CN108548920A (zh) * 2018-02-28 2018-09-18 江苏医诺万细胞诊疗有限公司 一种利用免疫磁珠负向吸附联合流式细胞法检测循环肿瘤细胞的试剂盒的检测方法
CN113832100A (zh) * 2021-10-26 2021-12-24 江苏大学附属医院 一种肿瘤肝转移组织免疫细胞的获取方法
WO2023224923A3 (fr) * 2022-05-16 2024-04-04 The Regents Of The University Of California Cellules modifiées et méthodes d'utilisation
CN119881336A (zh) * 2025-02-06 2025-04-25 北京青莲百奥生物科技有限公司 一种广泛兼容蛋白裂解液成分的蛋白定量试剂盒

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