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WO2019088445A1 - Puce de diagnostic de cellules immunitaires - Google Patents

Puce de diagnostic de cellules immunitaires Download PDF

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Publication number
WO2019088445A1
WO2019088445A1 PCT/KR2018/010975 KR2018010975W WO2019088445A1 WO 2019088445 A1 WO2019088445 A1 WO 2019088445A1 KR 2018010975 W KR2018010975 W KR 2018010975W WO 2019088445 A1 WO2019088445 A1 WO 2019088445A1
Authority
WO
WIPO (PCT)
Prior art keywords
immune
immune cell
immune cells
substance
unit
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/KR2018/010975
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English (en)
Korean (ko)
Inventor
양성
김한별
최종찬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gwangju Institute of Science and Technology
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Gwangju Institute of Science and Technology
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 Gwangju Institute of Science and Technology filed Critical Gwangju Institute of Science and Technology
Publication of WO2019088445A1 publication Critical patent/WO2019088445A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to an immune cell diagnostic chip for producing immune cells for enhancing the immune function of cancer patients by analyzing antigen responses of the immune cells based on immune cells of cancer patients.
  • Nanobiochip is a nanotechnology integrated with biochip. Nano-biochip technology combines nanotechnology (NT) and biotechnology (BT) to produce a variety of genes (DNA, RNA), proteins, cells and various metabolites on a chip By sensing directly with various sensors and various preprocessing functions, it is possible to diagnose diseases faster and more accurately, thereby contributing to the eradication of human diseases and quality improvement of healthy life.
  • This nano-biochip has advantages such as small size, low price, high performance, high integration, low power, relatively small amount of sample, fast reaction time and analysis time compared to micro-biochip. And is rapidly growing, expecting to make innovative changes in various fields such as life sciences, medicine, pharmacy, and clinical.
  • the present inventors have sought to develop a method of overcoming cancer by improving immune function in cancer patients.
  • an immune cell was collected from the cancer patient's body, and a chip was detected that detects immune cells effectively acting on the cancer of the cancer patient based on the collected immune cells.
  • a chip was detected that detects immune cells effectively acting on the cancer of the cancer patient based on the collected immune cells.
  • an immune cell diagnostic chip for analyzing an antigenic reaction of an immune cell based on immune cells of a cancer patient to enhance the immune function of the cancer patient
  • a method for producing immune cells comprising: a substrate; and a plurality of immune cell analysis units arranged on the substrate, wherein the immune cell analysis unit comprises: a capturing unit capturing the immune cells; And a detection unit for detecting the immunologically active substance.
  • the capturing unit may include an immobilization unit for immobilizing the immune cells to capture the immune cells.
  • the fixing unit may include a first fixing pin and a second fixing pin spaced apart from the first fixing pin.
  • the immune cells may be trapped between the first fixing pin and the second fixing pin.
  • the immune cells and the antigen are located in the capturing portion, and the interaction between the immune cells and the antigen can be performed.
  • the immunoactive substance is secreted by the interaction, and the immunoactive substance can move to the detection unit.
  • the detection unit can detect each of the plurality of substances.
  • the detection unit may include at least one protein marker that recognizes the immunoactive substance.
  • the detection unit may detect each of the plurality of substances by matching with any one of at least one protein included in the protein label.
  • the immunocyte analysis unit may include a plurality of detection units, wherein each of the plurality of detection units may be located adjacent to the capturing unit.
  • each of the plurality of detection units detects the immunoactive substance at a time interval, detects the immunoactive substance from any one of the plurality of detection units, and detects the immunoactive substance from another one of the plurality of detection units after a predetermined time interval
  • the secreted immunologically active substance can be detected.
  • the substance supply unit is configured to dissolve the immune cells and to immobilize a substance for amplifying the amount of remaining genes in the dissolved immune cells, To the analysis unit.
  • an immune cell diagnostic chip for producing an immune cell for enhancing the immune function of the cancer patient by analyzing the antigen response of the immune cell based on the immune cell of the cancer patient.
  • FIG. 1 is a plan view of an immune cell diagnostic chip according to an embodiment of the present invention.
  • FIG 2 shows an immunocyte analysis unit included in an immune cell diagnostic chip.
  • 3 is an exemplary illustration of the protein label of the detection unit included in the immune cell analysis unit.
  • FIG. 4 illustrates a detection process of the detection unit.
  • FIG. 5 shows dissolution of immune cells to analyze cell composition.
  • Figure 6 shows amplification of the amount of gene in dissolved immune cells and analysis of the base sequence.
  • Figure 7 shows the steps of capturing and analyzing immune cells in accordance with the present invention.
  • FIG. 1 is a plan view of an immune cell diagnostic chip according to an embodiment of the present invention
  • FIG. 2 is an immunocyte analysis unit included in an immune cell diagnostic chip
  • FIG. ≪ / RTI &gt is an immunocyte analysis unit included in an immune cell diagnostic chip
  • the immune cell diagnostic chip 10 of the present invention analyzes the antigenic reaction of the immune cells on the basis of the immune cells of a cancer patient to prepare an anti-cancer immune cell for enhancing the immune function of the cancer patient It is used in the method to do.
  • the immune cell diagnostic chip 10 may include a substrate 20, and a plurality of immune cell analysis units 100 arranged on the substrate 20.
  • the substrate 20 may be made of, for example, glass.
  • the immune cells collected from the cancer patient are captured and distributed in each of the immune cell analysis units 100 arranged on the substrate 20.
  • a plurality of immune cell analysis units 100 arranged on a substrate 20 of an immune cell diagnostic chip 10 includes a capturing unit 110 for capturing immune cells, And a detection unit 120 for detecting the secreted immunoactive substance.
  • a plurality of conductive patterns 101 of the immune cell analysis unit 100 can be formed.
  • the conductive pattern 101 may function as an electrode in the immune cell analysis unit 100.
  • the immunoactive substance secreted from the T-cell or the T-cell by the conductive pattern 101 can move to the neighboring conductive pattern 101.
  • the conductive pattern 101 is formed of a conductive material, and a magnet or a polymer pattern may be added.
  • the conductive pattern may be formed by a known patterning technique, such as a patterning method using a mask, or an inkjet printing technique.
  • the capturing unit 110 captures immune cells (T-cells) and immobilizes immune cells (T-cells) on the substrate 20.
  • the capturing unit 110 is included in each of the plurality of immune cell analysis units 100.
  • the immune cells (T-cells) are supplied from one side of the substrate 20, and the details thereof are beyond the scope of the present invention, and a detailed description thereof will be omitted.
  • the capturing unit 110 may include fixing units 111 and 112 for fixing immune cells (T-cells) to capture immune cells (T-cells) supplied from one side of the substrate.
  • the fixing parts 111 and 112 may be formed of a material such as a polymer material, a magnet material, or an optical structure.
  • the fixing parts 111 and 112 may include a first fixing pin 111 and a second fixing pin 112 to effectively capture the immune cells (T-cell).
  • the second fixing pin 112 and the first fixing pin 111 are spaced apart from each other.
  • the distance between the first fixing pin 111 and the second fixing pin 112 is the widest at the portion where the T-cell is introduced.
  • the distance between the first fixing pin 111 and the first fixing pin 111 increases as the distance from the inflow portion increases.
  • the second fixing pin 112 is narrowed.
  • the immune cells (T-cell) can be more effectively captured in the capturing part 110.
  • the immune cells (T-cell) are trapped between the first fixing pin 111 and the second fixing pin 112, and the distance between the first fixing pin 111 and the second fixing pin 112 is larger than the inflow width
  • the trapped immune cells (T-cell) can not pass between the first fixing pin 111 and the second fixing pin 112 and can be fixed on the capturing unit 110 have.
  • an antigen reacting with the immune cell is supplied to the capturing unit.
  • the antigen is a cancer antigen (neo antigen), which reacts with the receptor (TCR) of immune cells (T-cells). That is, the interaction between the T-cell receptor and the antigen can result in the interaction between the T-cell and the antigen.
  • TCR tumor receptor
  • the immunoactive substance secreted from the capture unit 110 by the T-cell moves to the detection unit 120.
  • At least one protein label 121 capable of recognizing an immunoactive substance is located in the detection unit 120 (see FIGS. 3 and 4).
  • the protein label 121 may include an antibody which is activated by an immunoactive substance.
  • the protein label 121 may include a plurality of proteins so that the detection unit 120 can detect each of the plurality of substances.
  • Each of the plurality of different substances is a substance capable of activating an antibody and can be matched with any one of a plurality of proteins contained in the protein label 121.
  • the immune cell analysis unit 100 may include a plurality of detection units 120 located adjacent to the capturing unit 110.
  • the immunoactive substance can be detected by the detection unit 120 at intervals of time. That is, at any one of the detecting units 120, the secreted immunoactive substance is detected at 0H (time). Thereafter, water is re-supplied to the capturing unit 110 to secrete the secreted secretions from the immune cells.
  • the solution contained an immunologically active substance.
  • the solution containing the newly generated immunoactive substance is transferred to the other detection unit 120 to detect the immunoactive substance after 6H. In this way, the immunologically active substance after 12H, 18H, 24H is detected. That is, the plurality of detection units 120 detect the immunoactive substance with a gap of time.
  • the above-mentioned time gap is exemplary only, and the present invention is not limited thereto.
  • an immune cell when an immune cell is bound to an antigen, it can be determined whether the immunologically active substance most effective for immunization is secreted. In other words, based on the antigen that the researcher can control, you can see which immune cells in the body react with the specific antigen. Based on these results, it is possible to design immune cells that are effective against cancers of cancer patients.
  • a plurality of substance supply units 300 may be positioned around the immune cell analysis unit 100 arranged on the substrate 20.
  • the substance supply unit 300 may supply the immune cell analysis unit 100 with, for example, an antigen (neo antigen), a washing solution, an antibody for detection, an immune cell lysate, a primer, a reverse transcriptase and the like.
  • the composition of the immune cells is analyzed by dissolving the immune cells.
  • the composition of the analyzed immune cells can be used to analyze antibodies that specifically react to specific antigens, and based on the analyzed data, the most effective antibodies can be tailored to cancer patients.
  • the amount of the gene remaining in the dissolved immune cells is amplified.
  • a known initiator and reverse transcriptase amplify the small amount of gene and analyze the base sequence.
  • immune cells necessary for cancer patients can be designed.
  • Immune cells are collected from the cancer patient's body. At this time, various kinds of immune cells can be collected. For example, dozens to thousands of species can be harvested.
  • each of the plurality of collected immune cells is fixed to each of a plurality of immune cell analysis units 100 included in the immunocellular diagnostic chip 10 described above. That is, each of the immune cell analysis units 100 can capture each of a plurality of collected immune cells (S10).
  • the immune cells and the antigen are reacted. More specifically, more than 20 kinds of antigens whose structures and characteristics have been identified are reacted with immune cells. At this time, the immune cells and antigens will react to each other. If it is a reaction, the immune cell will secrete an immunologically active substance. On the other hand, identification of immune cells including an antigen-bound receptor (TCR) is performed. Thereby, immune cells binding to a specific antigen can be identified (S20).
  • TCR antigen-bound receptor
  • the immunologically active substance secreted by the immune cell is detected, and when the immune cell is bound to an antigen, it can be known which secretes the immunologically active substance. Thereby, the characteristics of the antibody that specifically reacts with a specific antigen can be confirmed (S30).
  • composition of the immune cells is analyzed (S40).
  • the composition of the analyzed immune cells can be used to analyze antibodies that specifically react to specific antigens, and based on the analyzed data, the most effective antibodies can be tailored to cancer patients.
  • the amount of the gene remaining in the dissolved immune cells is amplified.
  • a small amount of the gene is amplified and the base sequence is analyzed (S50).
  • S50 base sequence
  • the present invention provides a method for detecting multiple immune cells from a cancer patient and simultaneously reacting multiple collected immune cells with the antigen to detect immune cells specifically reacting with a specific antigen, By designing the immune cells by manipulation, there is an effect of drastically improving the chemotherapy of cancer patients.
  • the number of immune cells reacting with an antigen, the kind and amount of each secreted protein in the immune cells, and the gene base sequence information of the immune cells can be seen at once, among many kinds of immune cells have.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
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  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

La présente invention concerne une puce de diagnostic de cellules immunitaires permettant de produire des cellules immunitaires afin d'améliorer la fonction immunitaire d'un patient atteint de cancer par l'analyse de réponses antigéniques de cellules immunitaires en fonction des cellules immunitaires du patient atteint de cancer. Une puce de diagnostic de cellules immunitaires de la présente invention comprend un substrat et une pluralité d'unités d'analyse de cellules immunitaires disposées sur le substrat, les unités d'analyse de cellules immunitaires pouvant comprendre une unité de capture permettant de capturer les cellules immunitaires et une unité de détection permettant de détecter un matériau immunoactif sécrété par les cellules immunitaires.
PCT/KR2018/010975 2017-11-03 2018-09-18 Puce de diagnostic de cellules immunitaires Ceased WO2019088445A1 (fr)

Applications Claiming Priority (2)

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KR1020170146021A KR20190050523A (ko) 2017-11-03 2017-11-03 면역세포 진단칩
KR10-2017-0146021 2017-11-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021179446A1 (fr) * 2020-03-13 2021-09-16 量准（上海) 医疗器械有限公司 Kit de dosage d'immunosorption de plasma numérique et procédés de fabrication et d'essai associés

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020120A1 (fr) * 1993-03-12 1994-09-15 Cellcor, Inc. Methode de titrage in vitro pour la mesure du degre d'activation de cellules immunes
KR20110010030A (ko) * 2009-07-23 2011-01-31 주식회사 해림후코이단 식품 선택을 위한 면역검사시스템
KR20110084118A (ko) * 2010-01-15 2011-07-21 이종균 개인별 맞춤형 면역증진식품의 선별을 위한 면역분석방법
KR20140093305A (ko) * 2013-01-08 2014-07-28 (주)아모레퍼시픽 피부 면역세포를 이용하는 피부 활성물질을 탐색하는 방법 및 이를 이용한 키트
KR20150130610A (ko) * 2014-05-13 2015-11-24 세종대학교산학협력단 T 보조 세포의 분화 방향성 측정용 아토피 피부염 동물모델 및 이를 이용한 약제 스크리닝 방법
KR20150145162A (ko) * 2014-06-18 2015-12-29 한국과학기술원 진단용 마이크로 칩 및 이를 이용한 통합형 회전식 진단 방법

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016196691A2 (fr) 2015-06-01 2016-12-08 California Institute Of Technology Compositions et procédés pour l'analyse de lymphocytes t avec des antigènes pour des populations spécifiques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020120A1 (fr) * 1993-03-12 1994-09-15 Cellcor, Inc. Methode de titrage in vitro pour la mesure du degre d'activation de cellules immunes
KR20110010030A (ko) * 2009-07-23 2011-01-31 주식회사 해림후코이단 식품 선택을 위한 면역검사시스템
KR20110084118A (ko) * 2010-01-15 2011-07-21 이종균 개인별 맞춤형 면역증진식품의 선별을 위한 면역분석방법
KR20140093305A (ko) * 2013-01-08 2014-07-28 (주)아모레퍼시픽 피부 면역세포를 이용하는 피부 활성물질을 탐색하는 방법 및 이를 이용한 키트
KR20150130610A (ko) * 2014-05-13 2015-11-24 세종대학교산학협력단 T 보조 세포의 분화 방향성 측정용 아토피 피부염 동물모델 및 이를 이용한 약제 스크리닝 방법
KR20150145162A (ko) * 2014-06-18 2015-12-29 한국과학기술원 진단용 마이크로 칩 및 이를 이용한 통합형 회전식 진단 방법

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021179446A1 (fr) * 2020-03-13 2021-09-16 量准（上海) 医疗器械有限公司 Kit de dosage d'immunosorption de plasma numérique et procédés de fabrication et d'essai associés

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