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WO2018107930A1 - Système de détection de cellules tumorales circulantes dans le sang périphérique et son application - Google Patents

Système de détection de cellules tumorales circulantes dans le sang périphérique et son application Download PDF

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WO2018107930A1
WO2018107930A1 PCT/CN2017/110403 CN2017110403W WO2018107930A1 WO 2018107930 A1 WO2018107930 A1 WO 2018107930A1 CN 2017110403 W CN2017110403 W CN 2017110403W WO 2018107930 A1 WO2018107930 A1 WO 2018107930A1
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peripheral blood
cancer
protein
tumor cell
magnetic beads
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Chinese (zh)
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江陆斌
尹世刚
景庆庆
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Institut Pasteur of Shanghai of CAS
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Institut Pasteur of Shanghai of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium

Definitions

  • the invention relates to the field of biomedicine, and more particularly to a peripheral blood circulation tumor cell detection system and application thereof.
  • cancer has the uncertainty of the location of the disease, the uncertainty of the pathogenesis, the early symptoms of the disease are not obvious, and the characteristics of high metastasis in the middle and late stages. Its prevention, detection and treatment have always been difficult for the medical profession. Solve the major problems. In the late stage of the disease, the infiltration of cancer cells is enhanced, the adhesion is weakened, and the cancer cells can escape from the solid tumor through the blood vessels, and further spread to other parts of the body by blood diffusion and form new lesions in appropriate new parts, thereby Causing effects, causing organ failure leading to individual death. The spread of cancer cells on a large scale is one of the important factors and signs of the deterioration of cancer.
  • Hematopoietic cancer cells also known as peripheral blood circulating tumor cells (CTC)
  • CTC peripheral blood circulating tumor cells
  • CTCs are a general term for various types of tumor cells present in peripheral blood. They are isolated from solid tumor lesions (primary tumors, metastases) into peripheral blood circulation due to spontaneous or diagnostic operations. Most CTCs undergo apoptosis or are phagocytized after entering the peripheral blood. A few can escape and anchor to develop into metastases, increasing the risk of death in patients with malignant tumors.
  • tumor metastasis is the leading cause of death in cancer patients.
  • Tumor cells invade into the surrounding tissues of primary tumor cells, enter the blood and lymphatic system, form circulating tumor cells CTC, and transfer to distant tissues, then ooze out, adapt to the new micro-environment, and finally "seeding" and “proliferation” , "planting” to form metastases ( Figure 1). Therefore, early detection of CTC in the blood has important guiding effects on patient prognosis, efficacy evaluation and individualized treatment.
  • academics have recognized the role of circulating tumor cells in revealing the metastatic behavior of tumors reflecting the invasiveness of tumors and evaluating the prognosis.
  • CellSearch detection method mainly uses cell automated capture instrument (System) uses a preset program and uses the CellSearch Cycle Tumor Cell Detection Kit ( Kit) can automatically and standardize samples for testing.
  • Tumor cells can be analyzed and counted using a CELLTRACKS II analyzer, a semi-automated fluorescence microscope. This count only counts the number of cells with epithelial cell characteristics (EpCAM + , CK8 + , CK18 + , and / or CK19 + ).
  • the CellSearch Circulating Tumor Cell Detection Kit includes a magnetic fluid capture reagent and a fluorescent immunoreagent.
  • a magnetic fluid reagent is a particle having a magnetic core. The surface is coated with an antibody recognizing the EpCAM antigen, which is an epithelial-derived CTC-specific antigen. Therefore, the magnetic particles can capture epithelial-derived CTCs.
  • Fluorescent reagents include the following components: cytokeratin antibodies (epithelial cell properties) specific for intracellular proteins against CK-phycoerythrin (PE); DAPI for nuclear staining; and anti-CD45-allophycocyanin (APC) Leukocyte-specific antibodies.
  • Reagent/sample mixture System assigned to an insert in The cells are presented in a cassette of the device. Said The device has a strong magnetic field that attracts magnetic particle-labeled epithelial cells on the surface of the cassette.
  • the CELLTRACKS Analyzer automatically scans the entire surface of the cassette, captures images and displays all events to the user, with CK-PE and DAPI fluorescent dye co-localized. The images are finally sorted and presented to the user in a gallery format. The events analyzed were classified as tumor cells when their morphological features were consistent with tumor cells and exhibited EpCAM + , CK + , DAPI + and CD45 - phenotypes.
  • the existing peripheral blood circulation tumor cell detection technology CellSearch has the following shortcomings:
  • This method can neither recognize epithelial cells with epithelial cell interstitial changes and tumor cell clusters in peripheral blood, nor recognize other tumor epithelial cells that do not express epithelial antigen EpCAM;
  • EpCAM EpCAM on the surface of some cancer cells is very low, which is not enough for sorting circulating tumor cells, such as liver cancer.
  • CTC detection methods target different tumor cell surface antigens. However, due to the variety of antigen types of different types of tumor cells, and more overlapping with normal cell antigens, such CTC detection methods are less specific. At present, there is no CTC test kit for targeting specific tumor cell surface antigens in China to obtain a new drug certificate.
  • the object of the present invention is to provide a peripheral blood circulation tumor cell detection system and application thereof.
  • a peripheral blood circulating tumor cell detection system comprising a buffer system and V2C coupled magnetic beads.
  • the buffer system comprises: glycerol, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and phosphate buffered saline (PBS), the buffer system having a pH of 7.0-7.6, preferably a pH of 7.2- 7.4.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • PBS phosphate buffered saline
  • the V2C coupled magnetic beads are a complex obtained by coupling a V2C protein or a variant thereof to a magnetic bead.
  • the glycerol is present in the buffer system at a level of from 3 to 8%, preferably from 4 to 6%, more preferably from 5%, based on the total volume of the buffer system.
  • the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the buffer system is from 10 to 50 mM, preferably from 20 to 30 mM.
  • the phosphate buffer comprises water, NaCl, KCl, Na 2 HPO 4 and KH 2 PO 4 .
  • the concentration of the Na 2 HPO 4 in the phosphate buffer is 4.0-4.5 mmol/L, preferably 4.2-4.3 mmol/L.
  • the concentration of the KH 2 PO 4 in the phosphate buffer is from 1.2 to 1.6 mmol/L, preferably from 1.3 to 1.4 mmol/L.
  • the concentration of the NaCl in the phosphate buffer is 130-140 mmol/L, and the concentration of the KCl is 2-3 mmol/L.
  • the buffer system is PBS.
  • the buffer system comprises: 7% BSA, 20 mM HEPES and water, the buffer system having a pH of from 7.0 to 7.6, preferably a pH of from 7.2 to 7.4.
  • the buffer system comprises: 10% BSA, 20 mM HEPES and water, the buffer system having a pH of 7.0-7.6, preferably a pH of 7.2-7.4.
  • the buffer system comprises: 0.5 glutaraldehyde, 20 mM HEPES and PBS, the buffer system having a pH of 7.0-7.6, preferably a pH of 7.2-7.4.
  • the V2C protein is derived from Plasmodium falciparum.
  • V2C protein is from the protein shown in NCBI Accession No. 811060.
  • V2C protein is from the protein shown in NCBI Accession No. 811060.
  • amino acid sequence of the V2C protein has the sequence set forth in SEQ ID NO:1.
  • amino acid sequence of the V2C protein is the sequence set forth in SEQ ID NO: 1.
  • the V2C protein variant has a sequence selected from the group consisting of SEQ ID NOs: 9, 14, 15, 18.
  • amino acid sequence of the V2C protein variant is selected from the group consisting of the sequences set forth in SEQ ID NOs: 9, 14, 15, 18.
  • the magnetic beads have a diameter of from 0.1 ⁇ m to 1 mm.
  • the magnetic beads are M-280 Tosyl activated (Tosylactivated) (Invitrogen, Catalog nos. 14203, 14204).
  • the magnetic beads are magnetic beads that can covalently couple amino and sulfhydryl groups.
  • the V2C protein or variant thereof is coupled to the magnetic beads by chemical methods.
  • the V2C coupled magnetic beads are prepared by: the V2C protein or a variant thereof The magnetic beads were incubated with the magnetic beads at 35-42 ° C for 12-36 h to obtain the V 2 C coupled magnetic beads.
  • the V2C protein or variant thereof is coupled to the surface of the magnetic beads in the form of a covalent bond.
  • the weight ratio of the magnetic beads to the V2C protein or variant thereof in the V2C coupled magnetic beads is 500: (0.1-10), preferably 500: (0.5-2).
  • the weight ratio of the magnetic beads to the V2C protein or variant thereof in the V2C coupled magnetic beads is (10-100): (0.1-10), preferably 50: (0.1-10).
  • the V2C coupled magnetic beads in the V2C coupled magnetic beads are stored at a concentration of 10-30 mg/ml, preferably 20 mg/ml.
  • the ratio of buffer system to V2C coupled magnetic beads in the detection system is 1 ml: (200-500) ⁇ g, preferably 1 ml: 400 ⁇ g.
  • the working concentration of the V2C coupled magnetic beads in the detection system is from 200 to 500 [mu]g/ml.
  • the buffer system and the V2C coupled magnetic beads in the detection system are each independent (separate storage).
  • the buffer system in the detection system is mixed with V2C coupled magnetic beads.
  • a peripheral blood circulating tumor cell test kit comprising the detection system of the invention.
  • the kit further comprises a monocyte separation solution Ficoll-Paque PLUS (GE Heathlthcare, Cat: 17-1440-03).
  • Ficoll-Paque PLUS GE Heathlthcare, Cat: 17-1440-03
  • the kit comprises:
  • a reaction system for detecting peripheral blood circulating tumor cells wherein the reaction system comprises the detection system of the present invention and a peripheral blood sample to be tested.
  • the volume ratio of the peripheral blood sample to the buffer system in the reaction system is (5-10):1, preferably 7.5:1.
  • the V2C coupled magnetic beads are stored in phosphate buffered saline (PBS) pH 7.5 at a concentration of about 10-30 mg/ml, preferably 20 mg/ml, to prepare a V2C coupled magnetic bead stock solution.
  • PBS phosphate buffered saline
  • the volume of the V2C coupled magnetic bead stock solution added per 5-10 ml of the peripheral blood sample is 10-30 ⁇ l.
  • the volume of the V2C coupled magnetic bead stock solution added per 7.5 ml of the peripheral blood sample is 20 ⁇ l.
  • the ratio of the peripheral blood sample to the V2C coupled magnetic beads in the reaction system is 7.5 ml: (200-500) ⁇ g, preferably 7.5 ml: 400 ⁇ g.
  • a detection system in the preparation of a detection reagent or detection kit for detecting peripheral blood circulating tumor cells.
  • the detection reagent or detection kit is for detection of a peripheral blood sample.
  • the source of the tumor cells includes epithelial cancer, stromal cancer, and hematopoietic cancer.
  • epithelial cancer includes lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast cancer, placental choriocarcinoma, cervical cancer, esophageal cancer, gastric cancer, liver cancer, ovarian cancer, colorectal cancer, prostate cancer, and pancreatic tumor.
  • stromal cancer includes rhabdomyosarcoma, osteosarcoma, and Ewing sarcoma.
  • hematopoietic cancer includes acute myeloid leukemia, multiple myeloma, B cell lymphoma, and T cell lymphoma.
  • the tumor cells comprise an epithelial cancer cell line, an stromal cancer cell line, and/or a hematopoietic cancer cell line.
  • the epithelial cancer cell line comprises lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast cancer, placental choriocarcinoma, cervical cancer, esophageal cancer, gastric cancer, liver cancer, ovarian cancer, colorectal cancer, prostate cancer And/or pancreatic tumors.
  • the mesenchymal cancer cell line comprises rhabdomyosarcoma, osteosarcoma, and/or Ewing's sarcoma.
  • the hematopoietic cancer cell line comprises acute myeloid leukemia, multiple myeloma, B cell lymphoma, and / or T cell lymphoma.
  • the concentration of circulating tumor cells in the peripheral blood is from 1 to 10/10 mL, preferably from 1 to 5/10 mL.
  • a fifth aspect of the invention there is provided a method of detecting peripheral blood circulating tumor cells, wherein the method comprises the steps of:
  • the peripheral blood sample is first mixed with the buffer system of the present invention to obtain a mixed solution, and then the V2C-coupled magnetic beads are added to the mixed solution.
  • the method is non-diagnostic, and more preferably, the method is a scientific method.
  • the source of the tumor cells includes epithelial cancer, stromal cancer, and hematopoietic cancer.
  • the epithelial cancer includes lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast cancer, placental choriocarcinoma, cervical cancer, esophageal cancer, gastric cancer, liver cancer, ovarian cancer, colorectal cancer, prostate cancer, and pancreatic cancer .
  • the stromal cancer comprises rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.
  • the hematopoietic cancer comprises acute myeloid leukemia, multiple myeloma, B cell lymphoma, and T cell lymphoma.
  • peripheral blood circulating tumor cell detection kit or detection reagent of the invention for detecting peripheral blood circulating tumor cells.
  • the source of the tumor cells includes epithelial cancer, stromal cancer, and hematopoietic cancer.
  • the epithelial cancer includes lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast cancer, placental choriocarcinoma, cervical cancer, esophageal cancer, gastric cancer, liver cancer, ovarian cancer, colorectal cancer, prostate cancer, and pancreas Tumor.
  • the stromal cancer comprises rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.
  • the hematopoietic cancer comprises acute myeloid leukemia, multiple myeloma, B cell lymphoma, and T cell lymphoma.
  • V2C protein variant selected from the group consisting of the amino acid sequences:
  • the V2C protein variant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-18.
  • the V2C protein variant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 14, 15, 18.
  • the V2C protein variant consists of the amino acid sequence set forth in SEQ ID NOs: 3-18.
  • the V2C protein variant consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 14, 15, 18.
  • nucleic acid molecule having a nucleic acid sequence encoding a V2C protein variant of the invention.
  • nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent conditions to a complement having a nucleotide sequence encoding a V2C protein variant of the invention, wherein the encoded polypeptide specifically recognizes p-CSA and / or the activity of placenta-like chondroitin sulfate glycosaminoglycan (pl-CSA).
  • a plasmid having the nucleic acid molecule of the invention having the nucleic acid molecule of the invention.
  • a vector having the nucleic acid molecule or plasmid of the invention.
  • a host cell comprising a V2C protein variant, nucleic acid molecule, plasmid, or vector of the invention.
  • a host cell for producing a V2C protein variant of the invention is provided.
  • the V2C protein variant, the nucleic acid molecule, the plasmid, the vector, or the host cell of the present invention are provided in the preparation of a peripheral blood circulation tumor cell detection system, or in the preparation of a peripheral blood circulation tumor cell detection kit. Or in the preparation of a reaction system for detecting circulating blood cells of peripheral blood.
  • FIG. 1 shows a schematic diagram of the spread of peripheral blood circulating tumor cells (CTC).
  • CTC peripheral blood circulating tumor cells
  • FIG. 2 shows the flow of the technical route of the present invention.
  • Figure 3 shows the results of the coupled magnetic beads of the present invention for capturing circulating tumor cells in blood samples of confirmed cases of gastric cancer.
  • Figure 4 shows the effect of the buffer system of five different components on the detection sensitivity.
  • the values in the graph are the average values after three independent experiments.
  • FIG. 5 shows the affinity of the V2C protein for tumor cells from different sources.
  • the values in the graph are the relative fluorescence intensity values of the V2C protein relative to the control protein.
  • Figure 6 shows the V2C-lacZ enzymatic reaction absorbance reading.
  • Figure 7 shows the amino acid sequence polymorphisms of different geographic strains V2C protein (Var2CSA).
  • Figure 8 shows the V2C-lacZ amino acid sequence in Example 4 of the present invention, the bold portion of the bold body is the V2C sequence, and the italic portion is the amino acid polymorphism region 546-836 involved (this region is used for artificial use in Example 4).
  • a candidate region for mutation which has a total of 15 polymorphic hotspots, and a C-terminal end of the sequence with a histidine tag for protein purification.
  • Figure 9 shows the relative absorbance values of V2C mutant - lacZ relative to V2C-lacZ.
  • V2C mutant- lacZ refers to: V2C protein variant is linked to ⁇ -galactosidase (abbreviated as ⁇ -gal) reporter gene;
  • V2C-lacZ refers to: V2C protein is linked to ⁇ -galactosidase reporter gene.
  • the inventors have extensively and intensively studied for the first time to unexpectedly find a peripheral blood circulation tumor cell detection system comprising a buffer system and V2C coupled magnetic beads.
  • the invention also provides V2C protein variants. Experiments have shown that with the buffer system and/or V2C protein variants of the invention, the efficiency and sensitivity of V2C coupled magnetic beads to capture circulating tumor cells can be significantly improved.
  • the buffer system of the present invention and the V2C coupled magnetic beads are used in combination to perform CTC detection, and have superior performance compared with the conventional CTC detection means.
  • the mutant V2C protein obtained by the method of artificially mutating a codon has a significantly improved affinity for cancer cells as compared with the V2C protein.
  • stringent hybridization conditions or “stringency” refers to about 5 ° C to about 20 ° C or 25 ° C below the melting temperature (Tm) of a target sequence and probe having an exact or near-accurate complement to the target.
  • Tm melting temperature
  • the melting temperature is the temperature at which a population of double-stranded nucleic acids becomes semi-decomposed into a single strand.
  • Tm 81.5 + 0.41 (% G + C) (see, for example, Anderson and Young, nucleic acid hybridization). Quantitative Filtration Hybridization (1985)). Other references include more complex calculations, considering structure and sequence features for calculating Tm.
  • stringent hybridization conditions are less than 1.0 moles per liter of sodium ion, typically from about 0.01 to 1.0 moles per liter of sodium ion at a salt concentration of from 7.0 to 8.3, a temperature of at least about 30 ° C for short probes, and at least about for long probes. 60 ° C (eg, greater than 50 nucleotides).
  • stringent conditions can also be obtained by the addition of destabilizing agents such as formamide, in which case lower temperatures can be utilized.
  • V2C protein Var2CSA
  • V2C protein The Plasmodium falciparum protein V2C protein (Var2CSA) can specifically recognize the polysaccharide structure on the surface of human cancer cells, and the normal cell surface of the human body does not have such a polysaccharide structure. Therefore, even the adjacent tissues can not bind to the protein, and the results show that the V2C protein is highly specific to cancer cells. At the same time, the V2C protein recognizes a wide range of cancer cells, covering almost all epithelial cancer cell lines, mesenchymal cancer cell lines and hematopoietic cancer cell lines, of which 96% of cancer cell lines (106 species) have affinity for V2C protein.
  • the V2C protein in the V2C protein or protein-coupled magnetic beads used is derived from the amino acid encoded by the gene in Plasmodium falciparum 3D7 (as set forth in SEQ ID NO: 1). The sequence is shown instead of the artificial variant protein of Example 4. See the appendix for a detailed sequence.
  • the magnetic beads used in the present invention are M-280 Tosyl activated (Invitrogen, Catalog nos. 14203, 14204).
  • the magnetic beads can covalently couple any material bearing an amino group and a thiol group to the surface of the magnetic beads without altering any biological properties of the protein. Since the proteins all contain an amino group, the foregoing M-280 Tosyl-activated magnetic beads can couple proteins to the surface of the magnetic beads in the form of covalent bonds (since the magnetic beads are commercially available reagents, please refer to the product specification for detailed features).
  • V2C coupled magnetic beads As used herein, the terms "V2C coupled magnetic beads”, “V2C protein coupled magnetic beads”, “Var2CSA protein-magnetic bead coupled complex VCMB”, “protein magnetic bead complex of the invention”, and “present invention”
  • the coupled magnetic beads are used interchangeably and refer to a complex (VAR2CSA-Magnetic Beads, VCMB) obtained by coupling a V2C protein (Var2CSA protein) to a magnetic bead.
  • the invention adopts the fusion protein expression technology to efficiently prepare the recombinant protein V2C protein in the prokaryotic expression system, and couples the protein with the magnetic beads, and successfully prepares the V2C protein-magnetic bead coupled complex VCMB of Plasmodium falciparum.
  • VCMB can not only identify a variety of cancer cells (epithelial cancer cell lines, stromal cancer cell lines and hematopoietic cancer cell lines), but also has a high positive detection rate (the detection limit of cancer cells is as low as 3). Cancer cells / 7.5 ml of blood), and the false positive rate is extremely low. It should be pointed out that this program has the advantages of detecting the most cancerous species, the highest specificity and the lowest false positive rate compared with the commercial CTC detection methods currently available on the market.
  • a special, non-human parasite protein is coupled to magnetic beads to form a protein magnetic bead complex VCMB.
  • the protein can be specifically used to combine the properties of placenta-like chondroitin sulfate glycosaminoglycan (pl-CSA) and p-CSA (placenta chondroitin glycosaminoglycan A) on the surface of cancer cells to make CTC rich. set.
  • the protein is derived from the erythrocyte membrane surface protein V2C expressed by Plasmodium falciparum.
  • the present invention has the following features:
  • VCMB can bind to many different types of cancer cells, such as the following cancers.
  • Epithelial cancer cell lines lung adenocarcinoma, lung squamous cell carcinoma, melanoma, breast cancer, placental choriocarcinoma, gastric cancer, liver cancer, cervical cancer, colorectal cancer, prostate cancer, esophageal cancer, ovarian cancer, pancreatic cancer, etc.;
  • hematopoietic cancer cell lines derived from the following cancers: acute myeloid leukemia, multiple myeloma, B-cell lymphoma, and T-cell lymphoma Wait.
  • the present invention is broader than the type of cancer cells recognized by EpCAM-mediated or other techniques of CTC.
  • VCMB-recognized ligands p-CSA and pl-CSA are polysaccharide structures that are only present on cancer cells and on the surface of maternal embryonic trophoblast cells. a polysaccharide-like form. Even the paracancerous tissues cannot bind to VCMB, which shows the high specificity of the protein-cancer cell interaction, and with this property, the protein magnetic bead complex of the present invention has a very strong targeting property.
  • VCMB can efficiently enrich tumor cells.
  • the highest sensitivity is up to 2.31 tumor cells per 7.5 ml sample.
  • buffer system and “buffer system” are used interchangeably and refer to a buffer system for the detection of peripheral blood circulating tumor cells.
  • a preferred buffer system comprises: glycerol, 4-hydroxyethylpiperazine ethyl sulfonate. Acid (HEPES), and phosphate buffered saline (PBS), and the pH of the buffer system is 7.0-7.6, preferably pH 7.2-7.4.
  • buffer systems useful in the present invention may be: 1) PBS; 2) 7% BSA, 20 mM HEPES and water, the pH of the buffer system being 7.0-7.6, preferably pH 7.2-7.4; 3) 10% BSA, 20 mM HEPES and water, the pH of the buffer system is 7.0-7.6, preferably pH 7.2-7.4; and 4) 0.5 glutaraldehyde, 20 mM HEPES, and PBS, the pH of the buffer system is 7.0-7.6, preferably The pH is 7.2-7.4.
  • the invention also provides a method for detecting peripheral blood circulation tumor cells comprising the buffer system.
  • the detection system further comprises V2C coupled magnetic beads. Wherein, the buffer system and the V2C coupled magnetic beads may be mixed or may be present separately.
  • the invention also provides a detection kit and a reaction system comprising the buffer system.
  • the inventors have tried a variety of different buffer systems, and it has been surprisingly found that different buffer systems have different efficiencies and sensitivities for the adsorption of V2C protein-coupled magnetic beads to different cancer cells.
  • the effect of sensitivity on the adsorption of cancer cells between the lines was between 4.32 and 6.75 times (Fig. 4).
  • the inventors found an optimal buffer system (glycerol 5%, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4), and under the buffer system, the V2C protein-magnetic bead coupling compound of the present invention was utilized.
  • the VCMB is subjected to CTC detection and subsequent research, and the present invention has more excellent performance than the current CTC detection means.
  • the invention also provides a method for detecting peripheral blood circulation tumor cells by using the detection system of the invention, the method comprising the steps of:
  • the microfluidic sorting system utilizes nanomaterial technology to simultaneously process a large number of samples in parallel, with high throughput, fast analysis speed, low loss, etc., and the required sample or reagent amount needs only a few microliters to several tens of microliters.
  • the microfluidic chip for detecting circulating tumor cells is customized according to the physical and biological characteristics of the circulating tumor cells, and the microfluidic device mainly includes a pressure device, an input device, a customized circulating tumor cell chip output device, and a detecting device.
  • Pre-treated peripheral blood samples (such as peripheral blood samples after removal of red blood cells by Ficoll) are added to the input device of the microfluidic system, the flow rate is controlled by a pressure device, and then the circulating tumor cells specifically enriched by the customized chip are collected. Can be used for downstream testing.
  • Circulating tumor cell test kit Due to the shortcomings of the existing circulating tumor cell detection technology, the inventors have improved their shortcomings, and developed a new generation of the new specific tumor targeting detection marker "V2C protein of Plasmodium falciparum (Var2CSA protein)". Circulating tumor cell test kit.
  • the reagents, kits and detection methods of the invention can be used in the following fields:
  • CTC Circulating Tumor Cell
  • Tumor/cancer big data collection Using captured CTCs, combined with current single-cell sequencing technology, we can build a detailed tumor/cancer database, build disease models, and support anti-tumor (cancer) development;
  • CTC can not only provide efficacy monitoring and prognosis, but also the molecular analysis of CTC can reflect the genetic information of patients' tumors and guide individualized medication.
  • the CTC detection system of the present invention is more sensitive, and the detection efficiency can reach 2.31 CTC/7.5 ml peripheral blood samples.
  • the CTC detection system of the present invention recognizes a wide variety of cancer cells with strong targeting specificity.
  • the CTC detection system of the present invention has a clear composition, contains no cytotoxic substances, and is easy to be popularized.
  • Tumor cell lines used in the experiment purchased from ATCC and Chinese Academy of Sciences cell bank
  • V2C gene and protein The pET-21a vector containing the V2C gene (Novagen, Cat. 69940-3) was constructed and stored in the laboratory in E. coli BL21 Rosetta-gami TM B (DE3), Novagen , Item No. 71136) was expressed and purified.
  • Ni 2+ affinity chromatography column GE company, article number 17524701.
  • RPMI 1640 Gbico article number 11875-085.
  • DAPI Life Technology item number P36971.
  • Buffer system which may contain water, bovine serum albumin (BSA), glutaraldehyde, 4-hydroxyethylpiperazineethanesulfonic acid (also known as hydroxyethylpiperazine ethanesulfate or HEPES), phosphate buffer (PBS), and / or glycerin, specifically including the following five formulations:
  • BSA bovine serum albumin
  • glutaraldehyde glutaraldehyde
  • 4-hydroxyethylpiperazineethanesulfonic acid also known as hydroxyethylpiperazine ethanesulfate or HEPES
  • PBS phosphate buffer
  • glycerin specifically including the following five formulations:
  • Formulation 1 phosphate buffered saline (PBS) (pH 7.4);
  • Formulation 2 BSA 7%, HEPES 20 mM (pH 7.4), water;
  • Formulation 3 BSA 10%, HEPES 20 mM, water, pH 7.4;
  • Formulation 4 5% glycerol, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4;
  • Formulation 5 0.5 ⁇ glutaraldehyde, HEPES 20 mM (pH 7.4), phosphate buffered saline (PBS), pH 7.4.
  • the concentration indicated in the scheme is the final concentration
  • the phosphate buffer solution contains NaCl 137 mmol/L, KCl 2.7 mmol/L, Na 2 HPO 4 4.3 mmol/L, and KH 2 PO 4 1.4 mmol/L.
  • the coding sequence is inserted into a suitable E. coli plasmid pET-21a, so that it can be recognized by the host expression system to synthesize the corresponding V2C protein.
  • V2C protein in E. coli by conventional protein synthesis systems in the art.
  • a high-efficiency protein synthesis system (based on GE's AKTATM fully automated protein purification system) built by the Shanghai Lushang Institute of the Chinese Academy of Sciences, based on the previous optimization strip.
  • the V2C gene in the plasmid was overexpressed in E. coli to obtain high levels of protein. According to the company's size, the protein production fermentation grade is determined.
  • V2C expressed protein from E. coli E.coli BL21Rosetta-gami TM B (DE3 ) (which transformed the expression plasmid pET-21a V2C protein) isolated, gets V2C pure protein.
  • Solution 4 (glycerol 5%, HEPES 20 mM, phosphate buffer (PBS), pH 7.4) is the most suitable buffer for optimal detection sensitivity and efficiency.
  • the V2C magnetic beads of the present invention can be sorted by conventional methods in the art, such as according to a commercially available magnetic stand (DynaMag-2 Magnet 12321D, thermofisher (Invitrogen)).
  • the present invention designs a microfluidic sorting instrument suitable for V2C magnetic beads, and cooperates with an experienced manufacturer of microfluidic instruments.
  • the CTC cells can be separated in the microfluidic sorting system by simply mixing the peripheral blood sample to be detected with the V2C magnetic beads.
  • the captured CTC can not be used conventional biological detection methods (microscopic detection, PCR (polymerase chain reaction) technology and immunolabeling ) for testing.
  • the present invention uses the most advanced single-cell sequencing technology in the world to detect and identify isolated cells. This method can not only qualitatively identify CTC cell types, but also obtain CTC cell gene layer by whole genome sequencing or transcriptome sequencing. More detailed information can provide the necessary reference for patients to provide follow-up targeted treatment programs.
  • Formulation 1 phosphate buffered saline (PBS) (pH 7.4);
  • Formulation 2 BSA 7%, HEPES 20 mM (pH 7.4), water;
  • Formulation 3 BSA 10%, HEPES 20 mM, water, pH 7.4;
  • Formulation 4 5% glycerol, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4;
  • Formulation 5 0.5 ⁇ glutaraldehyde, HEPES 20 mM (pH 7.4), phosphate buffered saline (PBS), pH 7.4.
  • Buffer Formulation IV is the optimal buffer formulation (glycerol 5%, HEPES 20 mM, phosphate buffer (PBS). ), pH 7.4), while Buffer Formula One (Phosphate phosphate buffer PBS, pH 7.4) is the worst-case buffer formulation in the selected formulation, and its detection effect is generally below 200 cells per 1000 initial cell.
  • Buffer Formulation IV has excellent detection efficiency, and It is about 4.32 times to 6.75 times that of the buffer one.
  • V2C proteins involved are all P. falciparum V2C proteins (as shown in SEQ ID NO: 1).
  • Plasmodium falciparum protein V2C specifically binds to tumor cells and demonstrates that not all Plasmodium falciparum proteins can specifically bind to tumor cells, highlighting the V2C protein specific and broad spectrum of Plasmodium falciparum Identify tumor cells.
  • the inventors incubated the recombinant control protein and the V2C protein with different tumor cells, respectively, and then performed the flow cytometry analysis using the antibody anti-6xHis tag-FITC (Abcam, Cat. No. ab1206).
  • the aforementioned recombinant control protein is a Plasmodium falciparum non-V2C protein containing a recombinant tag (the Plasmodium falciparum actin protein is used in the present embodiment).
  • the role of the aforementioned recombinant control protein in this example is to highlight that the Plasmodium falciparum protein V2C specifically binds to tumor cells.
  • the V2C protein is a V2C protein for magnetic bead coupling.
  • the C-terminus of the protein contains a recombinant tag, which can be used for experiments such as antigen-antibody binding, and the recombinant tag does not affect the affinity of the V2C protein and tumor cells.
  • V2C protein 100 ⁇ g was separately labeled with different cancer cells (H1792, AGS, SMMC7721, MNNG, MMG63, TC71, KG-1, NALM-6, MOLP-2, respectively, 1 million) in buffer formulation IV (glycerol 5 %, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4) After incubation for 30 min, the anti-6x His tag-FITC flow cytometry antibody was added for 60 min. The control group of each cell was incubated with the anti-6xHis tag-FITC antibody without V2C protein to indicate non-specific adsorption of the antibody to the cancer cells.
  • buffer formulation IV glycerol 5 %, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4
  • PBS phosphate buffered saline
  • the nuclear stain DAPI was added, and then analyzed by flow cytometry (BD company, model LSRFortessa), and 100,000 cells (ie, the control in FIG. 5) were counted, and the proportion of fluorescently labeled cells (ie, V2C) was counted. / control).
  • the blue column is the experimental group for each cancer cell, and the yellow column is the corresponding control group.
  • V2C protein has a strong affinity for tumor cells of different origins.
  • the specific experimental procedure is: adding a certain number of gradients (1, 5, 10, 20, 50, 100, and 1000) of cancer cells to healthy human red blood cells that remove leukocytes to simulate the tumor environment.
  • the V2C coupled magnetic beads were added to the simulated environment, and the mixture was uniformly shaken slowly. After 2 hours, the cells were enriched with a magnet holder, and the enriched cells were stained with DAPI and counted under a fluorescence microscope. The tests were repeated three times independently and the count was taken three times.
  • the limit detection rate is measured by a mixture of tumor cells and healthy human red blood cells, which mimics the distribution of tumor cells in human blood.
  • the specific experimental procedure is as follows: the mononuclear cell separation solution Ficoll-Paque PLUS removes most of the red blood cells from the peripheral blood samples of cancer patients, and then removes the white blood cells by CD45 + magnetic bead negative screening, and then thoroughly washes the remaining cells with phosphate buffered saline (PBS). Thereafter, it was fully suspended with Buffer Formulation IV (glycerol 5%, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4), and 50 mg of V2C coupled magnetic beads were added thereto for 2 hours at room temperature, and the magnetic beads were captured with a magnetic stand. The mixture was gently washed 5 times with the aforementioned buffer solution. DAPI stained cells were added. Cellular fluorescence was observed under a fluorescence confocal microscope. The experimental results show that in this experiment, the V2C coupled magnetic beads are very well enriched in the tumor cells of cancer patients, and the cell count indicates a good enrichment effect.
  • the buffer system used in the experiments involved in this example was the optimal buffer formulation obtained in Example 1 (ie, buffer formulation four: glycerol 5%, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4).
  • the V2C proteins involved were all P. falciparum V2C proteins (SEQ ID NO: 1).
  • SEQ ID NO: 1 P. falciparum V2C proteins
  • the specific experimental procedure was as follows: Mononuclear cell separation solution Ficoll-Paque PLUS was used to remove most of the red blood cells from healthy human peripheral blood samples, and the cells were thoroughly washed with phosphate buffered saline (PBS), and then buffered with IV (glycerol 5%, HEPES). 20 mM, phosphate buffered saline (PBS), pH 7.4) was fully suspended, and 50 mg of V2C coupled magnetic beads were added to incubate for 2 hours at room temperature, and the magnetic beads were captured with a magnetic stand. The mixture was gently washed 5 times with the aforementioned buffer solution. DAPI stained cells were added. Cellular fluorescence was observed under a fluorescence confocal microscope. The experimental results showed that there were almost no cells enriched in healthy humans in this experiment, indicating that the V2C-coupled magnetic beads have strong tumor cell specificity.
  • PBS phosphate buffered saline
  • IV glycerol 5%, HEP
  • Hepatoma cells HepG2 were adherently cultured in a 96-well plate to an area of about 80%, and washed 5 times with PBS buffer.
  • V2C-lacZ was added to buffer formulation IV (glycerol 5%, HEPES 20 mM, phosphate buffered saline (PBS), pH 7.4) to obtain a final concentration of 100 ng/ ⁇ l of detection solution.
  • the assay solution was added to 96 wells containing cells and incubated for 30 min at 25 °C. Rinse thoroughly 5 times with the aforementioned buffer formulation 4.
  • reaction buffer containing 400 ng/ml o-nitrobenzene ⁇ -D-galactopyranoside buffer formulation 4
  • o-nitrobenzene ⁇ -D-galactopyranoside is a galactosidase reaction
  • the substrate was prepared into a V2C-lacZ enzyme reaction system, and after fully reacting at 37 ° C, the reaction was terminated by adding 0.15 M Na 2 CO 3 , and the absorbance at a wavelength of 410 nm was detected in a microplate reader.
  • V2C-lacZ enzymatic activity was measured for different time periods within 30 min according to the methods (1) to (2) above.
  • the sample was taken out from the V2C-lacZ enzyme reaction system every 2 min, and the reaction was terminated with Na 2 CO 3 (Na 2 CO 3 was a stop solution of the galactosidase enzymatic reaction), and the absorbance at a wavelength of 410 nm was read.
  • Na 2 CO 3 was a stop solution of the galactosidase enzymatic reaction
  • V2C protein sequence Mutant sequence 1 QSKKNNKNW TSRSKKKWIWR 2 SSGKEG FPGKEG 3 CLVVCLDEKGKK YLGNLRKLENVC 4 QELKNIRTNS EDVTDINFDTK 5 LLKEWIIAA KFLAGCLIAA 6 PSHEKKNDDNGK TSHEKKNDDNGK 7 - NDDNNSK 8 NTAEQDTS IASDENTL 9 LAMKHGAGMNS IAMKHGAGMNG 10 TCCG TCSSGSGDNG 11 GSVTGSGSS GSVTGSSDSGST 12 - TCSGDNGSIS 13 ES NTSGERKI 14 KTECKNKCEV EKKCNKCEA
  • the amino acid polymorphism region (549-800) of the V2C gene of Plasmodium falciparum 3D7 was divided into 15 mutation hotspot regions (see Figure 7), wherein the sequence in column 1 of Table 3 was wild type in 3D7.
  • the amino acid sequence of V2C in the mutation hotspot region (1-15) we replace the corresponding column 1 sequence with the sequence of column 2 of the table.
  • the 3D7 strain does not have amino acid residues in the polymorphic hotspot regions 7 and 12 and is represented by "-".
  • V2C amino acid sequence 546-836 of different geographical strains of Plasmodium falciparum (Fig. 7), we divided the polymorphic region into 15 parts and sub-regionally mutated (the mutation method uses artificial synthesis containing mutated sequences).
  • the V2C gene fragment (546-836) was then amplified by a standard reverse PCR method using the pET-21a plasmid containing the V2C gene as a template, and the mutated fragment ligated with the DNA ligase to ligate the amplified fragment and the amplified vector.
  • V2C-lacZ to be mutated and substituted sequences are shown in Table 3.
  • the affinity of the mutated V2C-lacZ to HepG2 was detected by the method and conditions described in the step (3) of the present example, wherein the absorbance at 410 nm was detected after incubation for 20 min in the V2C-lacZ enzyme reaction system. Since the mutation in the V2C region only affects the affinity of V2C or its variant protein (V2C mutant ) with cancer cells, the lacZ activity does not change. Therefore, the numerical change in the enzyme activity reaction system is the expression of the affinity of V2C mutant and cancer cells.
  • V2Cmutant-lacZ proteins with different mutations (ie, lacZ linked to C-terminus, respectively, ligated variant proteins 1-15 (SEQ ID NOs: 3-17, respectively) and variant V2C (7+12+) 13)
  • the absorbance of the enzyme-active reaction system of the protein (the sequence of SEQ ID NO: 18) and the absorbance of the enzyme active system of the negative control group (V2C-lacZ protein, that is, the protein used in Example 4) were made.
  • ratio 1 indicates that the variant protein and affinity are the same, the ratio ⁇ 1 indicates that the variant protein affinity is weak, and the ratio > 1 indicates the variant
  • Protein affinity is stronger than V2C protein.
  • the nucleic acid sequence fragment (546-836) corresponding to the mutated sequence in this example was synthesized by Gene Synthesis Company (Shanghai Jieli Biotechnology Co., Ltd.).
  • the sequence containing no V2C (546-836) was amplified by reverse PCR using the vector originally expressing V2C-lacZ as a template, and the artificially mutated nucleic acid sequence was ligated to the vector by blunt-end ligation.
  • V2C amino acid sequence expressed by Escherichia coli in the present invention is shown in SEQ ID NO: 1, and the sequence is suitable for Examples 1-3, and is used as Example 4 after binding to LacZ at the C-terminus (SEQ ID NO: 2). In the comparison.
  • V2C-lacZ amino acid sequence of Example 4 of the present invention is shown in SEQ ID NO: 2.
  • variant protein 1 to variant protein 15 without lacZ and variant protein V2C (7+12+13) have amino acid sequences as shown in SEQ ID NOs: 3-18, respectively.
  • V2C amino acid sequence expressed by E. coli in the present invention is shown in SEQ ID NO: 1. This sequence was applied to Examples 1-3 and used as a control in Example 4 after binding to LacZ at the C-terminus (SEQ ID NO: 2).
  • the sequence of SEQ ID NO: 1 is as follows:
  • the amino acid sequence of V2C-lacZ in Example 4 of the present invention is shown in SEQ ID NO: 2, wherein the bolded part of the black body is a V2C sequence, and the italic part is the amino acid polymorphism region 546-836 involved (this region is an example).
  • a candidate region for artificial mutation in 4, which has a total of 15 polymorphic hotspots), and the sequence at the C-terminus of the sequence for the histidine tag for protein purification is SEQ ID NO: 2:
  • amino acid sequences substituted by amino acid polymorphism regions 546-836 are shown below as follows (a total of 16 human mutant sequences ( The V2C variant sequence), SEQ ID NO: 18 is a combination of three mutations): wherein the italic sequence is an amino acid polymorphic region and the italic bold sequence is an artificial mutant sequence.
  • Variant protein 1 (SEQ ID NO: 3):
  • Variant protein 2 (SEQ ID NO: 4):
  • Variant protein 3 (SEQ ID NO: 5):
  • Variant protein 4 (SEQ ID NO: 6):
  • Variant protein 5 (SEQ ID NO: 7):
  • Variant protein 6 (SEQ ID NO: 8):
  • Variant protein 7 (SEQ ID NO: 9):
  • Variant protein 8 (SEQ ID NO: 10):
  • Variant protein 9 (SEQ ID NO: 11):
  • Variant protein 10 (SEQ ID NO: 12):
  • Variant protein 11 (SEQ ID NO: 13):
  • Variant protein 12 (SEQ ID NO: 14):
  • Variant protein 13 (SEQ ID NO: 15):
  • Variant protein 14 (SEQ ID NO: 16):
  • Variant protein 15 (SEQ ID NO: 17):
  • V2C 7+12+13) (SEQ ID NO: 18):

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Abstract

L'invention concerne un système de détection de cellules tumorales circulantes (CTC) dans le sang périphérique. En particulier, l'invention concerne un système de détection comportant un système tampon et une bille magnétique conjuguée V2C ; en outre, un variant de protéine V2C est également décrit ; le système tampon préféré comporte : de la glycérine, de l'acide 4-hydroxyéthylpipérazine éthanesulfonique (HEPES), et une solution tampon de phosphate ; le pH dudit système tampon est compris entre 7,0 et 7,6, et un pH de 7,2 à 7,4 est préférable ; la bille magnétique conjuguée V2C est un complexe obtenu par conjugaison d'une protéine V2C ou d'un variant de cette dernière avec une bille magnétique. L'invention concerne également une trousse d'analyse et un système de réaction comportant ledit système tampon. Le système tampon et la bille magnétique conjuguée V2C sont utilisés en vue de la détection de CTC, et présentent une excellente efficacité par comparaison avec les moyens de détection de CTC existants.
PCT/CN2017/110403 2016-12-14 2017-11-10 Système de détection de cellules tumorales circulantes dans le sang périphérique et son application Ceased WO2018107930A1 (fr)

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