CN101182509A - Solid phase t cell selection used for antigenic specificity t cell purification - Google Patents
Solid phase t cell selection used for antigenic specificity t cell purification Download PDFInfo
- Publication number
- CN101182509A CN101182509A CNA2007101703216A CN200710170321A CN101182509A CN 101182509 A CN101182509 A CN 101182509A CN A2007101703216 A CNA2007101703216 A CN A2007101703216A CN 200710170321 A CN200710170321 A CN 200710170321A CN 101182509 A CN101182509 A CN 101182509A
- Authority
- CN
- China
- Prior art keywords
- cell
- basic unit
- cells
- antigen
- chloro
- 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.)
- Pending
Links
Images
Classifications
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
-
- 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
- C12N5/0081—Purging biological preparations of unwanted cells
- C12N5/0087—Purging against subsets of blood cells, e.g. purging alloreactive T cells
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/10—Mineral substrates
- C12N2533/12—Glass
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to solid phase T cell selection for antigen specific T cell purification. Aspects of the present invention relate to methods for binding cells to a substrate, removing target T cells from a population of T cells, depleting alloreactive T cells from a sample such as bone-marrow or stem cell transplant cells, and measuring the frequency of antigen-specific T cells in a sample.
Description
Invention field
The field of the invention comprises the solid phase t cell selection of fixing and be used for T cells with antigenic specificity purifying of cell in basic unit.The method of the frequency of T cells with antigenic specificity in aspect of the present invention relates to the method for removing with cell and basic unit's bonded method, with target T cell from the T cell mass, remove homogeneous reactivity T cell from the bone marrow transplantation cell method and the measure sample.
Background of invention
The adherent characteristic of attached cell has been used to several different methods, as based on microarray, organizational project and the separation of cell and the method for amplifying specific cell mass.Strengthen adherent character by the surface of using extracellular matrix protein (as collagen or fibronectin) or its deutero-peptide and polymer treatment such as the slide of gelatin bag quilt.Such method often depends on hydrophobic interaction.The main limitation of this class technology is its application to be extended to for example B cell that transforms of dendritic cell or EBV of non-adherent cell.In addition, depend on non covalent bond, interaction can cause the release of cell from basic unit.
Having developed multiple technologies is used for selection and amplification or removes T cells with antigenic specificity.The amplifying specific cell mass can strengthen it such as the treatment immune effect in the stem cell transplantation process, and wherein the patient can have benefited from for example increasing the lymphocyte populations that shows antitumous effect.It is also important that the method for from the group, removing the T cell.For example, from donor lymphocyte, remove homogeneous reactivity T cell to reduce the generation and the seriousness of potential fatal graft versus host disease (GVH disease).But described method application of difference density centrifugation, magnetic bead or associate with special peptide and with the fluorescence dye bonded MHC tetramer.These methods are expensive, consuming time usually, and efficient is low.Often lose antigen-specific.Other method is used the antigen presenting cell of elutriation, and as monocytic rete, but these methods are limited to attached cell, requires to know MHC restriction epitope peptide with selection antigen-specific sexual cell, and may require special whizzer (as Elutra
TM).
The frequency of measuring T cells with antigenic specificity in the given sample has several different methods.The most frequently used is that flow cytometry cell within a cell factor determination, ELISPOT measure and the MHC-tetramer is measured.These methods are measured the content (being concentration) of activatory antigen-specific cytotoxic t lymphocytes, but require special instrument, as ELISPOT reader or flow cytometry.These methods are consuming time and bothersome usually, and expensive.
The T cell therapy of adopting is studied as the method for malignant disease among the treatment mankind at present.To melanoma, nasopharyngeal carcinoma, Hodgkin lymphoma and leukemic clinical studies show result likely.It comprises T cell, the external activation of gathering immunology sensitization and these lymphocytes and these cells are introduced among the host again to cause disappearing of tumour of increasing clinically.Have multiplely be used to stimulate, the scheme of separation and stripped amplification effect cytotoxic T cell, each scheme is used the various combination of antigen presenting cell, cytokine, antigen form and concentration and thorn flyback cycle time length.Yet, have some clinical bottlenecks to limit its clinical application.Quantitative restriction (can not produce the antigen-specific CD8 T cell of sufficient amount) and restriction qualitatively (providing cytokine and common the stimulation to produce handicapped cytotoxic T cell owing to excessive) are provided for these.
For example, (4 to 5 months) make the special cytotoxic T cell of EBV of sufficient amount and quality to need the several months usually in the clinical trial of treatment nasopharyngeal carcinoma, Hodgkin lymphoma.In addition, the cost height that produces these T effector cells must make us stepping back, and does not allow its widespread use.
Summary of the invention
The present invention relates to be used for the solid phase t cell selection of T cells with antigenic specificity purifying.The method of the frequency of T cells with antigenic specificity in aspect of the present invention relates to the method for removing with cell and basic unit's bonded method, with target T cell from the T cell mass, remove homogeneous reactivity T cell from the sample such as marrow or stem cell transplantation cell method and the measure sample.
An aspect of of the present present invention relates to cell and basic unit's bonded method.In the method, basic unit is contacted with 3-aminopropyltriethoxywerene werene linking agent (silane linking agent), make the silane linking agent combine with basic unit.Then, with silane linking agent and 2-O-4,6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4, the contact of 6-two chloro-s-triazine linking agents (PEG linking agent) makes the PEG linking agent combine with the silane linking agent.Then PEG linking agent and cell are contacted under cell and the PEG linking agent bonded condition allowing.
The present invention relates on the other hand with cell and basic unit's bonded other method.In this method, with the basic unit and the 2-O-4 of albumen bag quilt, 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4, the contact of 6-two chloro-s-triazine linking agents makes linking agent combine with the basic unit of albumen bag quilt.Then, basic unit and linking agent and cell are being allowed cell and 2-O-4,6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4 contact under the 6-two chloro-s-triazine bonded conditions.
One skilled in the art will realize that employed basic unit can be glass, polymkeric substance, metal, metal alloy or any suitable basic unit in the different embodiments of the present invention.In addition, described basic unit can enough polymkeric substance, peptide or albumen bag quilt, and can be configured as sheet, pearl or other form easily.
In another embodiment, described basic unit is polyethylene terephthalate (PET) or glass with for example bovine serum albumin (BSA) or its segmental albumen bag quilt.
Another aspect of the present invention relates to the method that target T cell is removed from the T cell mass.In this method, antigen presenting cell is contacted with antigen, make antigen presenting cell offer antigen.Use any method of the present invention discussed above that antigen presenting cell is attached in the basic unit then.With the T cell mass with contact with basic unit bonded antigen presenting cell, make that the T cell to this antigen-specific combines with antigen presenting cell.Then will be to the non-specific T cell of antigen wash-out.
Another aspect of the present invention relates to the method that homogeneous reactivity T cell is removed from the cell sample such as marrow or stem cell transplantation cell.In this method, the antigen presenting cell that uses any method of the present invention discussed above will get autoreceptor combines with basic unit.Make donorcells pass through basic unit and with the antigen presenting cell of autoreceptor contacts under any donor T cell to this antigen-specific and the antigen presenting cell bonded condition allowing.Then with non-allogeneic reaction sexual cell from basic unit's wash-out.In one embodiment, described basic unit can be included in post or other the suitable vessel or container.
Another aspect of the present invention relates to the method for the frequency of T cells with antigenic specificity in the measure sample.At first, with antigen presenting cell with fluorochrome label for example, and with the T cell with the fluorochrome label that for example is different from the fluorescence dye that is used for the labelled antigen presenting cells.Then, use any method of the present invention discussed above that antigen presenting cell is fixing from the teeth outwards.Then antigen presenting cell is contacted with the antigen of being paid close attention to, makes antigen presenting cell offer this antigen, then, with lymphocyte join second surface and with two surface-mounted, make to form and buffy coats that the antigen presenting cell layer closely contacts.Then assemblage is hatched the sufficiently long time to allow immune cynapse formation.Then, the immune cynapse that forms is detected and counts.By measure the frequency of T cells with antigenic specificity divided by total lymphocyte count with the sum of the immune cynapse that forms.
In another embodiment,, and the T cell is used the fluorochrome label that for example is different from the fluorescence dye that is used for the labelled antigen presenting cells with for example fluorochrome label of antigen presenting cell, as mentioned above.Then with antigen presenting cell and T cell concentration and mix.Can form monolayer cell, these cells are on glass slide for example or for example between the glass slide.Then assemblage is hatched the sufficiently long time to allow immune cynapse formation.Then, the immune cynapse that forms is detected and counts.By measure the frequency of T cells with antigenic specificity divided by total lymphocyte count with the sum of the immune cynapse that forms.
Compared with former method, method of the present invention provides several advantages.In the method for former selection T cells with antigenic specificity, spontaneous adherent cell on the surface as monocyte, tends to float from the surface in time, and therefore is not used in selection albumen or tumor cell specific T cell.In addition, the selection that requires special whizzer to be used for elutriation monocyte or T cells with antigenic specificity of former method only limits to as MHC when to limit epitope peptide be known.In addition, unautogenous adherent antigen presenting cell on the surface, the B cell (LCL) that transforms as dendritic cell, EBV and other can be used to select T cells with antigenic specificity now.Those skilled in the art can approve that method of the present invention can be utilized the different cell of many kinds, include but not limited to those cells as herein described.
The T cell therapy of adopting is studied as the method for malignant disease among the treatment mankind at present.To melanoma, nasopharyngeal carcinoma, Hodgkin lymphoma and leukemic clinical studies show result likely.It comprises T cell, the external activation of gathering immunology sensitization and these lymphocytes and these cells are introduced among the host again to cause disappearing of tumour of increasing clinically.Have multiplely be used to stimulate, the scheme of separation and stripped amplification effect cytotoxic T cell, each scheme is used the various combination of antigen presenting cell, cytokine, antigen form and concentration and thorn flyback cycle time length.Yet, have some clinical bottlenecks to limit its clinical application.These comprise quantitative restriction (can not produce the antigen-specific CD8 T cell of sufficient amount) and limit (cytokine is provided and stimulates the handicapped cytotoxic T cell of generation altogether owing to excessive) qualitatively.
For example, (4 to 5 months) make the special cytotoxic T cell of EBV of sufficient amount and quality to need the several months usually in the clinical trial of treatment nasopharyngeal carcinoma, Hodgkin lymphoma.In addition, the cost height of producing these T effector cells must make us stepping back, and does not allow its widespread use.In order to overcome these obstacles, we have developed new solid phase t cell system of selection with purifying and expansion of antigen specific T-cells.In this method, the fixing agent of our development in laboratory is fixed on lymphoblastoid clone (LCL) on the surface of solid carrier, to select and purifying T cell.Basis hypothesis is that T cells with antigenic specificity is discerned it and closed associated antigen (cognate antigen), and with its combine faster than non-T cells with antigenic specificity.Our data proof are really more preferably selected T cells with antigenic specificity than non-T cells with antigenic specificity.After removing the buoyant cell, because the formation of immune cynapse " concentrates T cells with antigenic specificity " subsequently from the teeth outwards.Then by adding low dosage IL-2 (20U/ml) these activated T cells that increase.Uniting of selection and amplification makes us can produce purity＞60% and total cell＞10 in a week
8The LCL specific T-cells.Can reach the LCL specific T-cells that surpasses 90% purity after T cell selection that 2 weeks inherent second took turns and the amplification.When selecting the T cell with LCL, the LMP2 specific T-cells can be enriched to＞30%, this LCL is ballistic with the LMP2 epitope peptide.This new T cell purification method is not only carried out easily, and fast with cheap.
Brief description of drawings
Fig. 1 shows the synoptic diagram that carries out EBV specific T-cells purifying by the system of selection of solid phase t cell.
Fig. 2 is the figure that shows by the EBV specific T-cells purifying of solid phase t cell system of selection.
Fig. 3 is the figure that shows the influence of select time.
Fig. 4 shows that the T cell is selected and amplification Cytometric histogram afterwards.
Fig. 5 is showed cell toxicity test result's figure.
Fig. 6 shows the synoptic diagram that carries out the general approach of LMP2 specific T-cells purifying by the system of selection of solid phase t cell.
Fig. 7 demonstration is selected and the CLG specific T-cells dissolving of the generation ballistic T2 clone of CLG by the ballistic LCL of CLG, and for using CMV pp64 peptide P
495-503Ballistic T2 clone does not have significantly to be killed.Each point is represented the mean value of three repeated experiments data.
Fig. 8 shows that the lymphocytolysis that is produced by the different choice time is from body LCL, and when in LCL, adding MHC I antibody-like (w6/32), the remarkable percentage to K562 and LCL does not dissolve (as representative, showing from the cell toxicity data of selecting the T cell in 5 minutes).Each point is represented the mean value of three repeated experiments data.
The M27L specific T-cells that Fig. 9 demonstration is produced by fixed LCL system of selection is discerned its target and is dissolved melanoma cells with respect to unmatched monocyte preference.
Figure 10 shows that CEA CAP1-6D specific T-cells is selected and amplification.0.2% CAP1-6D specific T-cells is arranged before the selection.After selection for the first time and the amplification, the frequency of CAP1-6D specific T-cells increases to 10%.After selection for the second time and the amplification, the frequency of CAP1-6D specific T-cells increases to 35%.
Figure 11 shows that the WT1 specific T-cells is selected and amplification.0.2% WT1 specific T-cells is arranged before the selection.After selection for the first time and the amplification, the frequency of WT1 specific T-cells increases to 11%.After selection for the second time and the amplification, the frequency of WT1 specific T-cells increases to 40%.
The detailed description of invention
The method that the present invention relates to cell is combined with basic unit, with target T cell from the T cell mass The method of removing, homogeneous reactivity T cell is removed from the sample such as the bone-marrow transplantation cell Method and the method for measuring the frequency of T cells with antigenic specificity in the sample.
In one embodiment, the present invention relates to method that cell is combined with basic unit, the method Comprise following step: (i) provide basic unit; (ii) with described basic unit and 3-aminopropyl triethoxysilicane The contact of alkane bridging agent is so that bridging agent is combined with basic unit; (iii) with described basic unit and 3-aminopropan Ethyl triethoxy silicane alkane bridging agent and 2-O-4,6-two chloro-s-triazine-polyethylene glycol-2-O-4,6-dichloro The contact of-s-triazine, so that 2-O-4,6-two chloro-s-triazine-polyethylene glycol-2-O-4,6-two chloro-s-triazines Be combined with the APTES bridging agent; And (iv) and cell allow cell with 2-O-4,6-two chloro-s-triazine-polyethylene glycol-2-O-4 connect under the condition of 6-two chloro-s-triazine combinations Touch.
In another embodiment, the present invention relates to method that cell is combined with basic unit, the party Method comprises the following steps: that (i) provides albumen coated basic unit; (ii) basic unit that described albumen is coated With 2-O-4,6-two chloro-s-triazine-polyethylene glycol-2-O-4, the contact of 6-two chloro-s-triazine bridging agents makes Getting described bridging agent is combined with the basic unit that albumen is coated with; And (iii) with basic unit and bridging agent and cell Allowing cell and 2-O-4,6-two chloro-s-triazine-polyethylene glycol-2-O-4,6-two chloro-s-triazine combinations Condition under contact.
Basic unit is any dimensionally stable solid of wanting, and it can be by ceramic masses, glass Glass, metal, crystalline material, plastics, polymer or copolymer, its any combination or a kind of The coated composition of material on another kind of material. Such as, but not limited to (partly) noble metal as the gold or Silver; Glass material such as soda-lime glass, Pyrex glass, Vycor glass, quartz glass; Metal or Nonmetal oxide; Silicon, mono phosphoric acid ester ammonium salt and other such crystalline material; Transition metal; Plastics or polymer comprise dendritic, such as polyvinyl chloride, polyvinyl alcohol, poly-methyl Methyl acrylate, poly-(vinylacetate-maleic anhydride), poly-(dimethyl siloxane) monomethyl third Olefin(e) acid ester, polystyrene, polypropylene, polymine; Copolymer as poly-(vinylacetate-Altogether-maleic anhydride), poly-(styrene-altogether-maleic anhydride), poly-(ethene-altogether-acrylic acid) etc. Excellent Select in the embodiment, can modify basic unit realizing the fixing of biomolecule, such as, but not limited to, , formation patterned surface coated with gold or silver, etc.
Another embodiment of the present invention relates to the side that target T cell is removed from the T cell mass Method, described method comprises the following step: (i) antigen presenting cell is contacted with antigen, so that institute State antigen presenting cell and offer antigen; (ii) use any method of the present invention that described antigen is offered Cell binding is to basic unit; (iii) with T cell mass and the antigen presenting cell of being combined with basic unit Contact is so that be combined with antigen presenting cell to the T cell of this antigen-specific; And (iv) with right The T cell wash-out of this antigen non-specific.
Another embodiment of the present invention relates to be removed homogeneous reactivity T cell from transplanted cells The method of going, described method comprises: (i) use any method of the present invention will get the anti-of autoreceptor Former presenting cells is combined with basic unit; (ii) with donorcells by basic unit, thus with donorcells with The antigen presenting cell that gets autoreceptor is offered donor T cell and the antigen of antigen-specific in permission Contact under the condition of Cell binding; And (iii) with non-allogeneic reaction sexual cell from basic unit's wash-out.
Another embodiment of the present invention relates to the frequency of measuring T cells with antigenic specificity in the sample Method, described method comprises: (i) antigen presenting cell (APCs) is used fluorochrome label; (ii) with the T cell fluorochrome label that is different from for the fluorescent dye of APCs; (iii) make With any method of the present invention that APCs is fixing from the teeth outwards; (iv) with APCs with pay close attention to The antigen contact makes APCs offer antigen; (v) lymphocyte is joined second surface; (vi) With two surface-mounted so that form buffy coat with APCs layer close contact; (vii) Assemblage is hatched the sufficiently long time to allow immune cynapse to form; (viii) detect exempting from of forming The quantity of epidemic disease cynapse; And (ix) by using the immune cynapse sum that forms divided by TLC Measure the frequency of T cells with antigenic specificity. Perhaps, as mentioned above, antigen is carried Be the cell fluorochrome label, and with the T cell with being different from for the labelled antigen presenting cells The fluorochrome label of fluorescent dye. Then with antigen presenting cell and T cell concentration and mixed Be combined. Can form cell monolayer, these cells are between two sheet glass slides. So After assemblage is hatched the sufficiently long time to allow immune cynapse to form. Then, to forming The immunity cynapse detects and counts. By using the immune cynapse sum that forms divided by lymphocyte Sum is measured the frequency of T cells with antigenic specificity.
Embodiment
Embodiment 1-with cell fixation at glass surface or the surperficial material requested of polyethylene terephthalate (PET):
The salt solution of phosphate buffered (PBS) pH8.5
The PBS of pH2 (based on the 1N HCl coarse adjustment of the pH test paper of pH 0-14)
PBS
The pH test paper of pH0-14 (EM Science, cat.9590)
The pH test paper of pH6.5-10 (EMD Chemicals Inc, cat.9583)
The activated PEG polymkeric substance
Saturated sodium bicarbonate solution
The PBS solution of 5%BSA, aseptic
Principle: BSA can be spontaneous attached on glass or the pet sheet face.The activated PEG polymkeric substance has the activation functional group at its each end, and it can be connected on BSA and the cell surface protein.
Process:
The 5%BSA of 3ml is joined in the 100ml Pyrex beaker, and at room temperature place and spend the night.Incline and BSA solution and wash three times to remove the non-BSA that adheres to PBS.
5ml PBS is joined in the 50ml conical tube that contains 0.4 gram activated PEG polymkeric substance (pH can become acidity immediately).After the vigorous stirring, solution is joined with in the pretreated 100ml Pyrex beaker of BSA.Monitor pH to keep pH with the pH test paper 8 to 9.After room temperature (20 ℃) was through 15 minutes, inclining, and with PBS rinsing three times to remove remaining unreacted polymer.Add 3ml PBS (pH2).After adding acid PBS, can be immediately or after 24 hours with cell fixation from the teeth outwards.
The cell that is used for fixing of preparation: if indicate, irradiation cell at first before fixing.With 15 * 10
6The B cell (LCL) that EBV transforms or the cell of other type 50ml PBS washed twice.Not protein-contg cell suspension in 2.5ml PBS (pH8.5), and slowly is incorporated in the Pyrex beaker along inwall.Cover with aseptic lid, move at microscopically and observe.Do not disturb in 2-3 minute, make cell precipitate from the teeth outwards as early as possible.Then beaker is moved to cell culture incubator (37 ℃ and 5%CO
2).After 30 minutes, carefully shake and incline and substratum.Wash 3 times to remove the buoyant cell with 10%FCS/RPMI is careful.Allowing the fixed cell in incubator, in 10%FCS/RPMI 1640 or 10%NABS/RPMI1640, to recover 1 hour before the further experiment.
Result: 3.5 ± 0.2 * 10
6The LCL cell can be fixed on 100ml Pyrex
TmOn the surface of beaker.Fixing these back 24 hours 88 ± 2% cell survivals in the time of in being placed in cell culture incubator.
Embodiment 2-T cells with antigenic specificity is selected
At 75cm
2In the culturing bottle with 50 * 10 in the 50ml perfect medium (CM)
6Individual peripheral blood lymphocytes (PBMC) joins 5 * 10
6Among the LCL, described perfect medium is made up of RPMI 1640 substratum that contain 10% heat-inactivated normal AB serum.At the 7th day, these first activated lymphocytes are counted, and selected with the LCL cell that is fixed on the solid carrier surface.
Solid phase t cell chosen process:
The first round is selected: after LCL (with 8000rad irradiation) was fixed on the 100ml Pyrex beaker surface, the concentration that adds among the 3ml temperature CM was 25 * 10
6Ml
-1First activated lymphocyte (the 7th day).At 5,10 and 15 minutes examination select times.Remove non-adherent cell with transfer pipet.The attached cell of remainder is washed 3 times with warm CM is careful, and washing once more after hatching 15-30 minute.Behind the 24h, by removing attached cell, and it is transferred to (ultimate density 20Um1 among the 100ml CM of culturing bottle and the rhIL-2 with careful stirring of transfer pipet
-1).Added rhIL-2 in every 2-3 days.At the 14th day, analyze cultured cells by flow cytometry cell within a cell factor determination (ICC).Fig. 1 illustrates the general approach that the T cell is selected.
Second takes turns selection: be used to second at the 14th day by the T cell of selecting generation in 5 minutes and take turns selection of T cell and amplification.At the 21st day, analyze cultured cells by ICC.
Traditional E BV specific T-cells stimulating method
In contrast, we have also prepared polyclone EBV specific T-cells with traditional stimulating method.In culturing bottle with 50 * 10
6PBMC with carry out common cultivation from body LCLs (with 8000rad irradiation) with respondent/stimulator ratio of 10: 1.At the 7th day it is stimulated once more, stimulate once more at the 14th day respondent/stimulator ratio then with 4: 1.Since the 7th day, added in every then 2-3 days IL-2 to ultimate density be 20Uml
-1At the 21st day, analyze cultured cells by ICC.
The result: we use the PBMC from 3 donors to carry out T cell selection experiment, and some representational data presentation is as follows.
At the 7th day, in first activated lymphocyte, measure the CD8+T cell (donor 1, mean value 2%) that produces IFN-γ by cell within a cell factor determination (ICC) with LCL.Referring to Fig. 2.Select these lymphocytes (select time 5 minutes) with the LCL cell (■) that is fixed on the glass surface subsequently, and increase with IL-2 then.At the 14th day, in cultured cells, measure the CD8+T cell that produces IFN-γ by ICC.And then select these cultured cells, and increase with IL-2 then with the LCL cell (■) that is fixed on the glass surface.At the 21st day, in cultured cells, measure the CD8+T cell that produces IFN-γ by ICC.In contrast, identical first activated lymphocyte also stimulates with LCL, and does not carry out twice weekly selection (the 7th day and the 14th day) (▲).Behind IL-2 amplification T cell, measure the CD8+T cell that produces IFN-γ by ICC the 14th day (stimulating back 7 days for the first time) and the 21st day (stimulating back 7 days for the second time).
The influence of select time is referring to Fig. 3.At the 7th day, with the LCL cell selection identical first activated lymphocyte (donor 1) as shown in Figure 2 that is fixed on the glass surface.Select time is 5,10 and 15 minutes.In contrast, also stimulate these identical first activated lymphocytes, and do not select with respondent/stimulator ratio of 4: 1 with LCL.At the 14th day, measure the CD8+T cell that produces IFN-γ by cell within a cell factor determination (ICC).
Cell counting after selection of T cell and the amplification is referring to Fig. 4.As shown in Figure 3 first round T cell is selected and amplification (donor 1) after 7 days, by the Vi-cell counter to the T cell counting.
Cytotoxicity experiment is referring to Fig. 5.Can kill from body LCL by the T cell (donor 1) that solid phase t cell system of selection (select time 5 minutes) produces, but can not kill K562 clone.
Embodiment 3-LMP2 specific T-cells is selected
Process: do not had detectable LMP2 specific T-cells by LCL after first the activation at PBMC usually.And then stimulate first activated lymphocyte, and increase with IL-2 with LCL.
After LCLs (with 8000rad irradiation) is fixed on 100ml Pyrex beaker surface, attached cell is impacted 2h with the selected LMP2 epitope peptide of 10-20 μ M at 37 ℃.Remove substratum, and attached cell is washed 3 times in warm PBS.The concentration that adds then among the 3ml temperature CM is 25 * 10
6Ml
-1The lymphocyte of cultivation.Select time is 5 minutes.Remove non-adherent cell with transfer pipet.Remaining attached cell is washed three times with warm CM is careful, and washing once more after hatching 15-30 minute.Second day, by removing attached cell, and it is transferred to (ultimate density 20Uml among the 100ml CM of culturing bottle and the rhIL-2 with careful stirring of transfer pipet
-1).Added rhIL-2 in every 2-3 days.Select back the 7th day, analyze cultured cells by ICC.Fig. 6 illustrates the general approach that the T cell is selected.
Carry out LMP2 specific T-cells purifying by the system of selection of solid phase t cell, referring to Fig. 6.
Result: use 4 kinds of LMP2 peptides (all being limited to HLA-A*0201) in this experiment:
LLW：
LLWTLVVLL
CLG：
CLGGLLTMV
FLY：
FLYALALLI
TVC：
TVCGGIMFL
The frequency (mean value, donor 5) of LMP2 specific T-cells is before selecting:
LLW：0.8％
CLG：0.8％
FLY：0.8％
TVC：0.8％
Total LMP2-specific T-cells: 3.2%
Total EBV-specific T-cells: 15%
Select the frequency (mean value) of back LMP2 specific T-cells to be:
LLW：1.5％
CLG：4％
FLY：10％
TVC：7％
Total LMP2 specific T-cells: 22%
Total EBV-specific T-cells: 70%
Embodiment 4The structure of-people cellular segregation post
We have made up people's cellular segregation post with this new activated PEG polymkeric substance and granulated glass sphere.Wrap by granulated glass sphere with bovine serum albumin (BSA).Under condition mentioned above, add activatory PEG polymkeric substance then, make the PEG polymkeric substance be connected on the BSA.Adding LCLs according to condition mentioned above makes LCLs finally be combined on the granulated glass sphere.
This is in order to remove the T cell with the treatment malignant disease in human stem cell is transplanted.The purpose of removing the T cell is to reduce Acute GVHD (graft versus host disease (GVH disease)).Removing of T cell is to remove method by pearl, light at present, and it is very expensive and efficient is low.The cellular segregation post is made by receptor antigen presenting cells such as LCL or monocyte, and wherein these cells are fixed on the granulated glass sphere surface by activatory PEG polymkeric substance.Allow donor lymphocyte to flow through post.Those homogeneous reactivities T cell donor T cell of identification receptor antigen presenting cell (promptly can) can be selected and be retained in the post.Effusive cell can not cause GVHD from post.This process can significantly reduce cost and the workload that the T cell is removed.
Embodiment 5-carry out the LMP2 specific T-cells with fixed LCLs to select
At 75cm
2In the culturing bottle with 100 * 10 in the 50ml substratum
6Peripheral blood lymphocytes (HLA*A0201+) has EBV LMP2 peptide with load
CLG10 * 10 of GLLTMV (CLG)
6Monocyte activation, described substratum is made up of RPMI 1640 substratum that contain 10% heat-inactivated normal AB serum.At the 7th day, 0.8% CLG specific T-cells is arranged.
With (80Gy) of irradiation from body LCLs as
Embodiment 1Described fixing after, it was loaded 1 hour once more with peptide CLG, described from body LCLs before fixing with 2 μ M peptide CLG impact 4 hours.In cell culture incubator with activated lymphocytes (90 * 10
6± 10 in the 3mL substratum, 37 ℃) join in the fixed antigen presenting cell.Detected different select time (3,5,7.5,10 and 15 minutes).Terminal point at each select time is removed non-adherent cell.With the remaining attached cell of the careful washing of 37 ℃ substratum.37 ℃ hatch 30 minutes after repeated washing.At carrier surface, the cell that produces IFN-γ in the cell of selecting in 3,5,7.5,10 and 15 minutes is increased to 10%, 17%, 24%, 21% and 19% (mean value) respectively.After the T cell is selected second day and afterwards every other day by adding IL2 (20u/ml) the selected lymphocyte that increases then.At the 8th day, be that the cell that produces IFN-γ in 5,7.5 and 15 minutes the expanded cells is increased to 55%, 70% and 65% (mean value) respectively at select time.In being 5,7.5 and 15 minutes final expanded cells group, select time has 80 * 10 respectively
6, 100 * 10
6With 100 * 10
6(mean value) individual cell.These cell masses that surpass 90% (mean value) are CD3+CD8+.Cytotoxicity is killed and is shown in Fig. 7.
Embodiment 6-carries out the EBV specific T-cells with fixed LCLs and selects
At 75cm
2In the culturing bottle with 100 * 10 in the 50ml substratum
6Peripheral blood lymphocytes (HLA*A0201+) is with 10 * 10
6(80Gy) through irradiation activates from body LCLs, and described substratum is made up of RPMI 1640 substratum that contain 10% heat-inactivated normal AB serum.At the 7th day, 1.8% EBV specific T-cells is arranged.
Will through irradiation from body LCLs as
Embodiment 1Described fixing.In cell culture incubator with activated lymphocytes (90 * 10
6± 10 in the 3mL substratum, 37 ℃) join in the fixed antigen presenting cell.Detected different select time (1,3,5,10 and 15 minute).Terminal point at each select time is removed non-adherent cell.With the remaining attached cell of the careful washing of 37 ℃ substratum.37 ℃ hatch 30 minutes after repeated washing.At carrier surface, the cell that produces IFN-γ in the cell of selecting in 1,3,5,10 and 15 minute is respectively 22%, 44%, 20%, 12% and 8% (mean value).Then, second day after the T cell is selected and afterwards every other day by adding IL2 (20u/ml) the selected lymphocyte that increases.At the 7th day, be that the cell that produces IFN-γ in 3,5,10 and 15 minutes the expanded cells is increased to 80%, 65%, 50% and 45% (mean value) respectively at select time.In being 3,5,10 and 15 minutes final expanded cells group, select time has 30 * 10 respectively
6, 100 * 10
6, 120 * 10
6With 120 * 10
6(mean value) individual cell.When will be from the body monocyte with EBV LMP2 peptide
LLWTLVVLL (LLW), CLG and
FLYALALLI (FLY) impacts and when cultivating altogether by the lymphocyte of selecting and amplification is produced together, and the cell that produces IFN-γ in the lymphocyte that produces by selecting in 3,5,10 and 15 minutes has 17%, 19%, 9% and 7% (mean value) respectively.Here the lymphocyte that produces by the solid phase system of selection is discerned and is killed from body LCL, but K562 is not killed significantly; When adding MHC I antibody-like (w6/32) in LCL, this lymphocyte is not to killing (Fig. 8) significantly from body LCL yet.
Embodiment 7-carries out the melanoma specific T-cells with fixed LCLs and selects
At 75cm
2In the culturing bottle with 100 * 10 in the 50ml substratum
6Peripheral blood lymphocytes (HLA*A0201+) has 5 * 10 of M27L (Melan-A 26-35 variant ELAGIGILTV) with load
6Individual through the activation of (30Gy) of irradiation autologous dendritic cell, described substratum is made up of RPMI 1640 substratum that contain 10% heat-inactivated normal AB serum.At the 7th day, 0.8% M27L specific T-cells is arranged.
Will through (80Gy) of irradiation from body LCLs as
Embodiment 1Described fixing after, it was loaded 1 hour once more with peptide M27L, described from body LCLs before fixing with 10 μ M peptide M27L impact 4 hours.In cell culture incubator with activated lymphocytes (90 * 10
6± 10 in the 3mL substratum, 37 ℃) join in the fixed antigen presenting cell.Detected different select time (3,5,7.5 and 10 minutes).Terminal point at each select time is removed non-adherent cell.With the remaining attached cell of the careful washing of 37 ℃ substratum.37 ℃ hatch 30 minutes after repeated washing.At carrier surface, the cell that produces IFN-γ in the cell of selecting in 3,5,7.5 and 10 minutes is respectively 6%, 7.4%, 8% and 8% (mean value).Then, second day after the T cell is selected and every other day increase and selected 10 minutes lymphocyte afterwards by adding IL2 (20u/ml).At the 7th day, at the CD3+ cell (220 * 10 of establishing door
6,＞90% is the CD3+ cell, mean value) in produce IFN-γ cell be 65% (mean value).Referring to Fig. 9.
Embodiment 8-carries out CEA CAP1 6D specific T-cells with fixed LCLs and selects
There is the DCs (10 μ M) of CEA CAP1 6D to activate with load PBMCs (HLA*A0201+) at first,, has the CAP1-6D specific T-cells of 0.2% (mean value) then by after stimulating again with the ballistic monocyte of CEA CAP1 6D (10 μ M).Have the fixed LCL of CEA CAP1 6D (10 μ M) to carry out selecting the first time (10 minutes select times) with load, the IL2 amplification of using 20U/ml then existed 10 after 7 days
8(mean value) cell, wherein 10% (mean value) is CAP1-6D specific T-cells (Figure 10).There is the fixed LCL of CEA CAP1 6D (10 μ M) to carry out the T cell selection second time (10 minutes select times) with load in these lymphocytes, increased then 7 days.Have 10
8The cell of (mean value), wherein 35% (mean value) is CAP1-6D specific T-cells (Figure 10).
Embodiment 9-carries out the WT1 specific T-cells with fixed LCLs and selects
There is the DCs (10 μ M) of WT1 to activate with load PBMCs (HLA*A0201+) at first,, has the WT1 specific T-cells of 0.2% (mean value) then by after stimulating again with the ballistic monocyte of WT1 (10 μ M).Have the fixed LCL of WT1 (10 μ M) to carry out selecting the first time (10 minutes select times) with load, the IL2 amplification of using 20U/ml then existed 10 after 7 days
8(mean value) individual cell, wherein 11% (mean value) is WT1 specific T-cells (Figure 11).There is the fixed LCL of WT1 (10 μ M) to carry out the T cell selection second time (10 minutes select times) with load in these lymphocytes, increased then 7 days.Have 10
8The cell of (mean value), wherein 40% (mean value) is WT1 specific T-cells (Figure 11).
Can make various modifications or variation to the present invention obviously to those skilled in the art, and not deviate from the spirit and scope of the present invention.Therefore the present invention is intended to cover modifications and variations of the present invention, condition be its drop on claims and the scope that is equal in.
Claims (20)
1. with cell and basic unit's bonded method, described method comprises:
A., basic unit is provided,
B. described basic unit and 3-aminopropyltriethoxywerene werene are contacted under 3-aminopropyltriethoxywerene werene and the described basic unit bonded condition allowing,
C. with described basic unit and the 2-O-4 of step b, 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4,6-two chloro-s-triazines are allowing 2-O-4, and 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4 contact under 6-two chloro-s-triazines and the 3-aminopropyltriethoxywerene werene bonded condition; And
D. described basic unit and the cell with step c allowing described cell and 2-O-4, and 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4 contact under the 6-two chloro-s-triazine bonded conditions.
2. with cell and basic unit's bonded method, described method comprises:
A., the basic unit of albumen bag quilt is provided,
B. with the basic unit and the 2-O-4 of described albumen bag quilt, 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4,6-two chloro-s-triazines are allowing 2-O-4, and 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4 contact under basic unit's bonded condition of 6-two chloro-s-triazines and described albumen bag quilt; And
C. described basic unit and the cell with step b allowing cell and 2-O-4, and 6-two chloro-s-triazine-polyoxyethylene glycol-2-O-4 contact under the 6-two chloro-s-triazine bonded conditions.
3. the method that target T cell is removed from the T cell mass, described method comprises:
A. antigen presenting cell is contacted with antigen, makes described antigen presenting cell offer antigen,
B. use method as claimed in claim 1 or 2 that described antigen presenting cell is attached in the basic unit,
C. under the described antigen presenting cell bonded condition that allows the T cell of the antigen-specific of step a and step b, contacting T cell mass and step b with the described antigen presenting cell of basic unit's bonded; And
D. will be to the T cell wash-out of the antigen non-specific of step a.
4. the method that homogeneous reactivity T cell is removed from sample, described method comprises:
A. the antigen presenting cell that uses method as claimed in claim 1 or 2 will get autoreceptor combines with basic unit,
B. make the sample that comprises donorcells described basic unit by step a, thus with described sample and step a the antigen presenting cell of autoreceptor contacts under from the antigen presenting cell bonded condition to the T cell of the antigen-specific of step a and step a in the described sample in permission; And
C. with non-allogeneic reaction sexual cell from described basic unit wash-out.
5. the method for the frequency of T cells with antigenic specificity in the measure sample, described method comprises:
A. with antigen presenting cell (APCs) with the first marker mark,
B. the T cell is used the second marker mark of described first marker that is different from step a,
C. use method as claimed in claim 1 or 2 that the APCs of step a is fixed on the first surface,
D. the APCs with step c contacts with the antigen of being paid close attention to, and makes described APCs offer antigen,
E. lymphocyte is added to second surface,
F. with described two surface contacts, make the lymphocytic layer of step e form and closely contact with the layer of the APCs of steps d, the formation assemblage,
G. the described assemblage of step f being hatched the sufficiently long time forms to allow immune cynapse,
H. detect the number of the immune cynapse that forms; And
I. by measure the frequency of T cells with antigenic specificity divided by total lymphocyte count with the immune cynapse sum that forms.
6. the method for the frequency of T cells with antigenic specificity in the measure sample, described method comprises:
A. with antigen presenting cell (APCs) with the first marker mark,
B. the T cell is used the second marker mark of described first marker that is different from step a,
C. with the T cytomixis of the mark of the APCs of the mark of step a and step b,
D. the described mixture with step c contacts with first surface,
E. the described surface with steps d covers with second surface, forms assemblage,
F. the described assemblage of step e being hatched the sufficiently long time forms to allow immune cynapse,
G. detect the number of the immune cynapse that forms; And
H. by measure the frequency of T cells with antigenic specificity divided by total lymphocyte count with the immune cynapse sum that forms.
7. as the described method of claim 1-6, wherein said basic unit is selected from glass, plastics, metal, metal alloy, pottery or polymkeric substance.
8. as the described method of claim 1-6, wherein with polymkeric substance or albumen bag by described basic unit.
9. as the described method of claim 1-6, wherein said basic unit is a pearl.
10. method as claimed in claim 8 is wherein wrapped by the albumen of described basic unit and is comprised BSA.
11. method as claimed in claim 1 or 2, wherein said cell comprise antigen presenting cell, tumour cell and by the cell of pathogenic infection.
12. as the described method of claim 1-6, wherein said cell is a non-adherent cell.
13. as the described method of claim 1-6, wherein said basic unit is a glass slide.
14. method as claimed in claim 11, wherein said antigen presenting cell are non-adherent cell.
15. as the described method of claim 1-6, wherein said basic unit comprises polyethylene terephthalate.
16. as the described method of claim 1-4, wherein said basic unit is in post.
17. method as claimed in claim 3, wherein said donorcells comprises marrow or peripheral stem cell.
18. method as claimed in claim 4, wherein said donorcells comprises marrow or peripheral stem cell.
19. method as claimed in claim 5, wherein described first marker of step a comprises first fluorescence dye, and described second marker of step b comprises second fluorescence dye.
20. method as claimed in claim 6, wherein described first marker of step a comprises first fluorescence dye, and described second marker of step b comprises second fluorescence dye.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US85828706P | 2006-11-10 | 2006-11-10 | |
| US60/858,287 | 2006-11-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101182509A true CN101182509A (en) | 2008-05-21 |
Family
ID=39402426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101703216A Pending CN101182509A (en) | 2006-11-10 | 2007-11-12 | Solid phase t cell selection used for antigenic specificity t cell purification |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101182509A (en) |
| WO (1) | WO2008061047A2 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6326083B1 (en) * | 1999-03-08 | 2001-12-04 | Calipher Technologies Corp. | Surface coating for microfluidic devices that incorporate a biopolymer resistant moiety |
| US6660523B2 (en) * | 2000-09-21 | 2003-12-09 | Schering Corporation | Dendritic cells; methods |
-
2007
- 2007-11-12 CN CNA2007101703216A patent/CN101182509A/en active Pending
- 2007-11-12 WO PCT/US2007/084385 patent/WO2008061047A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008061047A2 (en) | 2008-05-22 |
| WO2008061047A3 (en) | 2008-11-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2022001069A (en) | Antigen-presenting cell mimetic scaffold, and method for fabricating and using the same | |
| de la Rosa et al. | Interleukin‐2 is essential for CD4+ CD25+ regulatory T cell function | |
| CN106687584B (en) | Soluble antibody complexes for T cell or NK cell activation and expansion | |
| AU2002240818C1 (en) | MHC molecule constructs and their uses for diagnosis and therapy | |
| CN100490895C (en) | CD40-binding APC-activating molecules | |
| JP4117031B2 (en) | Purification of antigen-specific T cells | |
| Lea et al. | Characterization of human mononuclear cells after positive selection with immunomagnetic particles | |
| Geppert et al. | Activation of human T4 cells by cross-linking class I MHC molecules. | |
| Sarnacki et al. | Enhancement of CD3‐induced activation of human intestinal intraepithelial lymphocytes by stimulation of the β7‐containing integrin defined by HML‐1 monoclonal antibody | |
| AU633940B2 (en) | Method for stimulating proliferation of peripheral blood lymphocytes | |
| BR112016024072B1 (en) | in vitro method of stimulating a cell population | |
| CN108474791A (en) | Cultivate the method for cell and kit and equipment for this method | |
| Vujanovic et al. | Distinct phenotypic and functional characteristics of human natural killer cells obtained by rapid interleukin 2-induced adherence to plastic | |
| CN101243187A (en) | Method of producing lymphocytes | |
| EP2495312B1 (en) | Method for producing antigen-specific b cell population | |
| JPH04506061A (en) | CD8-based formulations | |
| CA2141428A1 (en) | Methods for positive immunoselection of stem cells | |
| Berger et al. | CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy | |
| CN101041816B (en) | Artificial antigen presenting cell and preparation method thereof | |
| Oberg et al. | An optimized method for the functional analysis of human regulatory T cells | |
| CN101182509A (en) | Solid phase t cell selection used for antigenic specificity t cell purification | |
| Mullins et al. | Transfection of thyroid autoantigens into EBV-transformed B cell lines. Recognition by Graves' disease thyroid T cells. | |
| Kondo et al. | Requirements for the functional expression of OX40 ligand on human activated CD4+ and CD8+ T cells | |
| Darling et al. | In vitro immune modulation by antibodies coupled to tumour cells | |
| CN112430575A (en) | Universal CAR-T cell, preparation method and application thereof, and antitumor drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080521 |