CN105504042A - Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application - Google Patents
Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application Download PDFInfo
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- CN105504042A CN105504042A CN201610079872.0A CN201610079872A CN105504042A CN 105504042 A CN105504042 A CN 105504042A CN 201610079872 A CN201610079872 A CN 201610079872A CN 105504042 A CN105504042 A CN 105504042A
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
Abstract
The invention relates to the field of biotechnology, in particular to a capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, a method for detecting the content of the cells, and application. The catching probe comprises ligands capable of being combined with the CAR-T cells, and fluorescence molecules combined on the ligands. The method comprises the following steps: adding the ligands combined with the fluorescence molecules into a sample to be detected, and carrying out mixing, so as to ensure that the ligands are combined on the CAR-T cells of the sample to be detected; carrying out centrifugation, and removing supernatant to reserve precipitates; resuspending the precipitates, and carrying out flow detection, so as to obtain the content of the CAR-T cells. The capture probe and the method can be used for fast and accurately realizing detection of the CAR-T cells, and providing powerful guarantee for monitoring and prognosis of the CAR-T cells.
Description
Technical field
The present invention relates to biological technical field, detect the capture probe of CAR-T cell, the detection method of this cell content and application in particular to a kind of.
Background technology
CAR-T, i.e. ChimericAntigenReceptorT-CellImmunotherapy, full name is Chimeric antigen receptor T cell immunotherapy.CAR-T cell (Chimeric antigen receptor T cell) be in the antigen-binding portion of antibody by certain tumour antigen can be identified and the born of the same parents of CD3-ζ chain or Fc ε RI γ part in vitro coupling be a chimeric protein, by the T cell of the method transfection patient of gene transfer, it is made to express Chimeric antigen receptor.
The T cell of patient, by after " recodification ", generates the specific CAR-T cell of massive tumor.Similar with other immunotherapy, its ultimate principle is exactly utilize the immunocyte of patient self to remove cancer cells, but unlike, this is a kind of cell therapy, instead of a kind of medicine.
More specifically, in the process implemented, CAR-T treats flow process and comprises collection peripheral blood in patients, is separated T cell, CAR imported T cell, quality control and cell is fed back to patient.Wherein, CAR is one section of mosaic gene, is a transmembrane protein after expression.The extracellular region of CAR expression product is the one section of sequence being derived from monoclonal antibody district, can identify cancer cell antigen, thus ensures that T cell is implemented to remove accurately to target cell.When after CAR-T cell recognition target cell, the intracellular region of CAR expression product can the killing activity of acute activation T cell, and then causes target cell apoptosis and cracking.
For CAR-T therapy, in recent years, it has shown splendid result for the treatment of in multiple clinical trial, is the cancer therapy new way very with potentiality.In a clinical trial for refractory acute lymphoblastic leukemia, CAR-T therapy makes the experimenter of 90% (27/30) obtain complete incidence graph (NEnglJMed2014; 371:1507-17.).At another for being grown up in the clinical trial of refractory acute lymphoblastic leukemia, CAR-T therapy makes the experimenter of 88% (14/16) obtain complete incidence graph (ScienceTranslationalMedicine2014; 6:224).
In addition, dozens of is also had for the CAR-T therapy of the diseases such as multiple myeloma, lymphoma, lung cancer, liver cancer, mammary cancer just in clinical trial.In future, CAR-T therapy is expected to become the important method of several late cancers treatment, clinical meaning and marketable value huge.
But the elimination efficiency that CAR gene can be expressed, the activity of the height of expression efficiency, CAR-T cell and purity all directly can affect CAR-T cells on cancer cells.Clinical study proves, the multiplication capacity after CAR-T cell feeds back in peripheral blood in patients and curative effect have very strong dependency (ScienceTranslationalMedicine2015; 7:303).
At present, the clinical trial of main flow adopts the CAR content in the method detection peripheral blood genome of real-time fluorescence quantitative PCR, and then indirectly estimates CAR-T cell quantity.But problem is, only have the CAR competence exertion effect of successful expression, and CAR content in genome is not directly related with the expression level of CAR.In addition, the copy number of CAR in genome and vector properties, virus titer, cell state etc. have relation, can result through the error of the CAR content estimation CAR-T cell detected in peripheral blood genome further.Therefore, detect CAR content accurately and rapidly, for the enforcement of this therapy, monitoring and prognosis, there is critical significance.
In view of this, special outstanding the present invention.
Summary of the invention
The object of the present invention is to provide a kind of capture probe for detecting CAR-T cell, large by the error existing for CAR content in detection genome and then indirect estimation CAR-T cell quantity to solve in prior art, the inaccurate technical problem of detected result.
The second object of the present invention is to provide a kind of method utilizing this capture probe flow cytometer detection CAR-T cell content, slow to solve existing detection method detection speed, the insecure technical problem of result; The method can realize the detection of CAR-T cell rapidly and accurately, for CAR-T monitoring, prognosis provide powerful guarantee.
The third object of the present invention is to provide the purposes of above-mentioned capture probe, to realize it at detection CAR-T cell content, for the preparation of the using value detected in the product (as corresponding test kit) of CAR-T cell content.
In order to realize above object, the technical solution used in the present invention is as follows:
The invention provides a kind of capture probe detecting CAR-T cell, described capture probe comprises the part that can be combined in CAR-T cell, and is combined in the fluorescence molecule on this part.
In the present invention, capture probe mainly comprises and can identify and be combined in the part of CAR-T cell; Because this part can be combined on CAR-T cell effectively, be specially and be attached to CAR molecule and (should understand, CAR molecule is herein the expression product of CAR gene) scFv structural domain on, and then the effect of catching CAR-T cell can be played, on the other hand because this part can also be combined with fluorescence molecule, and then can rapidly and accurately to CAR-T cell quantification by drain cell instrument.
By this capture probe, the direct quantitative that can realize CAR-T cell detects, and by preferred part, increases the combination rate of itself and CAR molecule, thus realizes the detection by quantitative of CAR-T cell quickly and accurately; (the CAR content in genome is not directly related with the expression level of CAR with traditional detection by CAR gene contained in genome, and only have the CAR competence exertion effect of successful expression) and indirectly estimate that the mode of CAR-T cell is compared, the estimation error that the factors such as vector properties, virus titer, cell state that also directly avoid may cause.
Preferably, described part comprises the first part and Ligands;
Described first part can be combined on CAR molecule scFv structural domain; And the first part can also be combined with vitamin H; Described Ligands can identify described vitamin H, and described fluorescence molecule can be combined on described Ligands.
In above-mentioned scheme, further refinement is carried out to part, first part its can be combined on CAR molecule scFv structural domain, but also can stably by biotin labeling, and Ligands identifiable design vitamin H, thus play the effect of catching, and Ligands can be marked by fluorescence molecule, to realize streaming detection by quantitative.
Preferably, described first part comprises the albumen that can be combined on lightchain or heavychain of IgG; And/or described Ligands is can in conjunction with the albumen of vitamin H or polypeptide, described fluorescence molecule comprises any one in FITC, PE, PE-CYx, APC.
Preferably, described first part comprises the variant of ProteinL, ProteinA, ProteinG and this several albumen; And/or described Ligands is Streptavidin.
In such scheme, by preferred ligandin, fluorescence molecule thus realize capture probe with detected cell better in conjunction with effect, to improve the accuracy of detection further.
Wherein, ProteinL, ProteinA, ProteinG and their variant, all can be combined on lightchain or heavychain of IgG, therefore can be incorporated on the scFv structural domain of CAR molecule; Streptavidin can effectively in conjunction with avidin molecules such as vitamin Hs.
Based on a method for flow cytometer detection CAR-T cell content, comprise the following steps:
Mixing adding the part being combined with fluorescence molecule in sample to be checked, making on the CAR-T cell of ligand binding in sample to be checked; Then centrifugal and supernatant discarded, retains throw out; Carry out flow cytometer detection after throw out is resuspended, obtain CAR-T cell content.
In detection method, the part being combined with fluorescence molecule in advance mixes on the rear CAR-T cell combined in the sample (being specially on the scFv structural domain of CAR molecule) with sample to be checked, when added part q.s, it substantially can by the successful all CAR-T cell captures of expression CAR, centrifuged deposit thing can carry out flow cytometer detection through resuspended, and then can know the content of CAR-T cell in sample rapidly and accurately.
Preferably, described method specifically comprises the following steps:
1), in streaming damping fluid sample to be checked is mixed with the first part being combined with vitamin H, centrifugal in conjunction with carrying out first time after 15-45 minute in 2-8 DEG C, obtain the first throw out;
2), by after the first throw out washing utilize streaming damping fluid resuspended, then add the Ligands being combined with fluorescence molecule, and centrifugal in conjunction with carrying out second time after 15-45 minute in 2-8 DEG C, obtain the second throw out;
3), by centrifugal after the second throw out washing, and flow cytometer detection is carried out after utilizing streaming damping fluid resuspended gained precipitation.
In above-mentioned concrete detection method, specific selection has been carried out to the temperature and time in different integrating step, and resuspended before carried out washing operation, thus improve the purity of target substance in precipitation, increase the accuracy of detected result.
Preferably, in step 2) and step 3) in, described washing all adopts streaming damping fluid to carry out.
Preferably, described first time, centrifugal centrifugal force was 500-1000g, and the time is 5-10min; The centrifugal centrifugal force of described second time is 500-1000g, and the time is 5-10min.
In centrifugal process, selection for centrifugal condition is vital, while suitable centrifugal speed and temperature can ensure solid-liquid separation effect, also ensure cyto-architectural integrity, prevent from selecting improper due to temperature and speed and damaging cellularstructure.
Capture probe described in claim is detecting the application in CAR-T cell content.
The application of capture probe described in claim in the product for the preparation of detection CAR-T cell content.
In view of the technique effect that this capture probe can reach; it is detecting CAR-T cell content and also ought to belong within protection scope of the present invention, such as, according to the test kit for detecting CAR-T cell content that this capture probe is made for the preparation of the application detected in the product of CAR-T cell content.
Compared with prior art, beneficial effect of the present invention is:
(1), this capture probe and the content of CAR-T cell in sample to be checked can be realized in conjunction with the method based on flow cytometer detection CAR-T cell content that this probe is carried out rapidly and accurately, whole detection method is easy and simple to handle, for the enforcement of CAR-T therapy, monitoring and prognosis provide powerful guarantee.
(2), this capture probe is applied to CAR-T cell detection and after preparing corresponding testing product, effectively can promotes the enforcement of CAR-T therapy, help the mankind and capture cancer early.
Accompanying drawing explanation
In order to be illustrated more clearly in the specific embodiment of the invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the detected result of vitro culture CAR-T cell provided by the invention;
Fig. 2 is the detected result after patient provided by the invention feeds back CAR-T.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly; clear, complete description is carried out below by technical scheme of the present invention; based on the embodiment in the present invention; other embodiments all that those of ordinary skill in the art obtain under the prerequisite not making creative work, all belong to the scope that the present invention protects.
Embodiment 1
The present embodiment provide for detecting the capture probe of CAR-T cell and utilizing this capture probe as follows based on the method for flow cytometer detection CAR-T cell content:
S11: the capture probe detecting flow cytometer detection CAR-T cell is provided;
This capture probe comprises the ligandin (ProteinL) in conjunction with CAR (expression product), and this protein binding has fluorescent tag molecule FITC (fluorescein isothiocyanate).
S12: cell to be detected is provided, this cell expressing CAR to be detected;
This cell to be detected is vitro culture CAR-T cell.
S13: mix adding ligandin in cell sample to be checked in streaming damping fluid, makes on the CAR-T cell of ligand binding in sample to be checked;
S14: by centrifugal for the product of step S13 and supernatant discarded, retains throw out, and throw out is resuspended after carry out flow cytometer detection.
Embodiment 2
The present embodiment provide for detecting the capture probe of CAR-T cell and utilizing this capture probe as follows based on the method for flow cytometer detection CAR-T cell content:
S21: the capture probe detecting flow cytometer detection CAR-T cell is provided;
This capture probe comprises the first ligandin ProteinL in conjunction with CAR (expression product), and this albumen is marked with vitamin H; In addition, the fluorescence molecule (PE, phycoerythrin) also comprising the Ligands albumen (Streptavidin) that can identify vitamin H and be marked on this Streptavidin.
S22: cell to be detected is provided, this cell expressing CAR to be detected;
In this step, measuring samples is prepared by the following method:
1) from peripheral blood, isolate mononuclearcell (PBMC);
2), from PBMC, T cell is sub-elected;
3), utilize slow-virus transfection system to carry out genetic modification to T cell, prepare the different algebraically CAR-T of multiple different target spot.
S23: in streaming damping fluid, sample to be checked is mixed with the ProteinL being combined with vitamin H, centrifugal in conjunction with carrying out first time after 30 minutes in 4 DEG C, obtain the first throw out;
S24: utilize streaming damping fluid resuspended by after the first throw out washing, then add the Streptavidin being combined with phycoerythrin, and centrifugal in conjunction with carrying out second time after 30 minutes in 4 DEG C, obtain the second throw out;
S25: by centrifugal after the second throw out washing, and carry out flow cytometer detection after utilizing streaming damping fluid resuspended gained precipitation.
In the above-described embodiment, the molecular structure of vitamin H is as follows:
Embodiment 3
The present embodiment provide for detecting the capture probe of CAR-T cell and utilizing this capture probe as follows based on the method for flow cytometer detection CAR-T cell content:
S31: the capture probe detecting flow cytometer detection CAR-T cell is provided;
This capture probe comprises the first ligandin (ProteinA) in conjunction with CAR (expression product), and this albumen is marked with vitamin H; In addition, the Ligands albumen (Streptavidin) that can identify vitamin H and the fluorescence molecule APC be marked on this Streptavidin is also comprised.
S32: cell to be detected is provided, this cell expressing CAR to be detected;
Consistent with step S22, do not repeat at this.
S33: in streaming damping fluid, sample to be checked is mixed with the ProteinA being combined with vitamin H, centrifugal in conjunction with carrying out first time after 40 minutes in 8 DEG C, obtain the first throw out;
S34: utilize streaming damping fluid resuspended by after the first throw out washing, then add the Streptavidin being combined with APC, and centrifugal in conjunction with carrying out second time after 40 minutes in 8 DEG C, obtain the second throw out;
S35: by centrifugal after the second throw out washing, and carry out flow cytometer detection after utilizing streaming damping fluid resuspended gained precipitation.
Embodiment 4
The present embodiment provide for detecting the capture probe of CAR-T cell and utilizing this capture probe as follows based on the method for flow cytometer detection CAR-T cell content:
S41: the capture probe detecting flow cytometer detection CAR-T cell is provided;
This capture probe comprises the first ligandin (ProteinL) in conjunction with CAR (expression product), and this albumen is marked with vitamin H; In addition, the Ligands albumen (Streptavidin) that can identify vitamin H and the fluorescence molecule PE-CY be marked on this Streptavidin is also comprised
5.
S42: cell to be detected is provided, this cell expressing CAR to be detected;
Consistent with step S22, do not repeat at this.
S43: in streaming damping fluid, sample to be checked is mixed with the ProteinL being combined with vitamin H, centrifugal in conjunction with carrying out first time after 45 minutes in 2 DEG C, obtain the first throw out;
S44: utilize streaming damping fluid resuspended by after the first throw out washing, then add and be combined with PE-CY
5streptavidin, and centrifugal in conjunction with carrying out second time after 45 minutes in 2 DEG C, obtain the second throw out;
S45: by centrifugal after the second throw out washing, and carry out flow cytometer detection after utilizing streaming damping fluid resuspended gained precipitation.
Embodiment 5
The present embodiment provide for detecting the capture probe of CAR-T cell and utilizing this capture probe as follows based on the method for flow cytometer detection CAR-T cell content:
S51: the capture probe detecting flow cytometer detection CAR-T cell is provided;
This capture probe comprises the first ligandin (ProteinL) in conjunction with CAR (expression product), and this albumen is marked with vitamin H; In addition, the Ligands albumen (Streptavidin) that can identify vitamin H and the fluorescence molecule FITC be marked on this Streptavidin is also comprised.
S52: cell to be detected is provided, this cell expressing CAR to be detected;
This cell to be detected is vitro culture CAR-T cell, and the concrete following steps that adopt realize:
From peripheral blood, isolate mononuclearcell (PBMC), and sub-elect T cell from PBMC; Utilize Retroviral Transfer system to carry out genetic modification to T cell, prepare multiple different target spot different algebraically CAR-T cell.
S53: in streaming damping fluid, sample to be checked is mixed with the ProteinL being combined with vitamin H, centrifugal in conjunction with carrying out first time after 35 minutes in 4 DEG C, obtain the first throw out;
S54: utilize streaming damping fluid resuspended by after the first throw out washing, then add the Streptavidin being combined with fluorescein isothiocyanate, and centrifugal in conjunction with carrying out second time after 30 minutes in 4 DEG C, obtain the second throw out;
S55: by centrifugal after the second throw out washing, and carry out flow cytometer detection after utilizing streaming damping fluid resuspended gained precipitation.
In the above-described embodiment, first time, centrifugal centrifugal force was 800g, and centrifugation time is 8min; The centrifugal centrifugal force of second time is 800g, and centrifugation time is 6min.
Embodiment 6
The present embodiment provide for detecting the capture probe of CAR-T cell and utilizing this capture probe as follows based on the method for flow cytometer detection CAR-T cell content:
S61: the capture probe detecting flow cytometer detection CAR-T cell is provided;
Consistent with the capture probe that embodiment 5 provides.
S62: cell to be detected is provided, this cell expressing CAR to be detected;
This cell to be detected is the peripheral blood sample after vitro culture CAR-T cell and patient CAR-T feed back.
S63: mixed with the ProteinL being combined with vitamin H by sample to be checked in streaming damping fluid, carried out first time centrifugal (500-800g, 5-10min) in 4 DEG C, obtains the first throw out after 35 minutes;
S64: by the first throw out with centrifugal after streaming buffer solution, use streaming damping fluid resuspended again, then add the Streptavidin being combined with fluorescein isothiocyanate, and after 35 minutes, carry out centrifugal (500-800g once again in 4 DEG C, 5-10min), the second throw out is obtained;
S65: by the second throw out with centrifugal after streaming buffer solution, and carry out flow cytometer detection after utilizing streaming damping fluid resuspended gained precipitation, the wavelength of flow cytometer detection looks the kind setting of fluorescence dye.
In addition, it is pointed out that in all of the embodiments shown above, the aminoacid sequence of ProteinL and the aminoacid sequence table of Streptavidin are respectively as shown in SEQIDNO:1 and SEQIDNO:2.
Application examples 1
The capture probe provided with embodiment 6 and its detection method detect the CAR-T cell sample cultivated through external structure, arrange the culture sample of control group, the 7th day, the 8th day and the 10th day respectively.Detected result as shown in Figure 1.
As seen in Figure 1, the CAR-T cell of vitro culture, along with the growth of incubation time, its CAR-T cell content increases continually and steadily.
In this application examples, concrete preparation and the cultural method of the CAR-T cell sample of external structure cultivation are as follows:
1) monocytic preparation
(1:1) in the peripheral blood of collection is joined with pipette, extract DPBS or physiological saline, diluted blood cell, hemocyte diluent is slowly added in the centrifuge tube that lymphocyte separation medium (Ficoll or Histopaque-1077) is housed, after the centrifugal 20-30 of 800g minute, draw the tunica albuginea confluent monolayer cells above lymphocyte separation medium, proceed in a new centrifuge tube, add Lonzax-vivo15 serum-free cell culture medium, supernatant is abandoned after centrifugal, retain the cell precipitation bottom centrifuge tube, namely obtain Cord blood mononuclear cells.
2) by the enrichment from the monocyte in Cord blood source of CD3 positive cell
The Cord blood mononuclear cells obtained is counted, adds the beads of Ou Lian CD3/CD28 antibody according to the ratio of 1:1, shake 20min gently, utilize magnetic frame, obtain the cell of the CD3 positive.
3) CAR-T is prepared
By step 2) in the CD3 positive cell that obtains utilize Lonzax-vivo15 substratum (containing the cytokines such as IL-2 and deactivation AB blood plasma) to cultivate.After 48 hours, the slow virus containing CAR is utilized to infect cultured cells with MOI (20-50).After 12 hours, carry out full dose and change liquid, continue to cultivate.
4) CAR-T quality control
When prepared by CAR-T culturing process and cell product, quality control need be carried out to it, comprise following several aspect: 1. detection of mycoplasma; 2. chlamydozoan detects; 3. intracellular toxin detects; 4. Bacteria Detection; 5. fungal detection; 6. viral residue detection; 7. CAR expression rate; 8. T cell nature examination; 9. Cytotoxicity in vitro ability detects.
Application examples 2
The capture probe provided with embodiment 6 and the content of its detection method to the CAR-T cell feeding back patient detect, and detect sample and arrange control group respectively, feed back 8 days samples.Detected result as shown in Figure 2.
As can be seen from Figure 2, after feeding back CAR-T8 days, CAR-T cell contained in patient body comparatively control group is compared, and CAR-T cell content increases obviously.
CAR-T therapy is when applying, and patient accepts the also potential clinical risk-cytokine storm of CAR-T therapy.Cytokine storm is also cytokines release syndrome.Producing reason is that T cell is killing other cell, such as can discharge a lot of albumen (cytokine) when bacteriophage, they can activate more immunocyte and carry out enantiopathy substance together, and this positive feedback mechanism ensure that the quick removing to pathogenic agent.
But due to CAR-T killing cancer cell be too fast too effective really, so moment super amount cytokine can be produced in local, and cause surprising immune response (clinical manifestation is sometimes exactly patient's high fever), if control bad, then there is very large risk in patient.Therefore by this fast and effeciently detection CAR-T cell content provided by the invention, (direct object is not the state of health in order to obtain diagnostic result or patient, but obtain intermediate information) method, can well for the clinical monitoring of patient.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. for detecting the capture probe of CAR-T cell, it is characterized in that, described capture probe comprises the part that can be combined in CAR-T cell, and is combined in the fluorescence molecule on this part.
2. capture probe according to claim 1, is characterized in that, described part comprises the first part and Ligands;
Described first part can be combined on CAR molecule scFv structural domain; And the first part can also be combined with vitamin H; Described Ligands can identify described vitamin H, and described fluorescence molecule can be combined on described Ligands.
3. capture probe according to claim 2, is characterized in that, described first part comprises the albumen that can be combined on lightchain or heavychain of IgG;
And/or described Ligands is can in conjunction with the albumen of vitamin H or polypeptide, described fluorescence molecule comprises any one in FITC, PE, PE-CYx, APC.
4. capture probe according to claim 3, is characterized in that, described first part comprises the variant of ProteinL, ProteinA, ProteinG and this several albumen;
And/or described Ligands is Streptavidin.
5. based on a method for flow cytometer detection CAR-T cell content, it is characterized in that, comprise the following steps:
Mixing adding the part being combined with fluorescence molecule in sample to be checked, making on the CAR-T cell of ligand binding in sample to be checked; Then centrifugal and supernatant discarded, retains throw out; Carry out flow cytometer detection after throw out is resuspended, obtain CAR-T cell content.
6. the method for detection CAR-T cell content according to claim 5, it is characterized in that, described method specifically comprises the following steps:
1), in streaming damping fluid sample to be checked is mixed with the first part being combined with vitamin H, centrifugal in conjunction with carrying out first time after 15-45 minute in 2-8 DEG C, obtain the first throw out;
2), by after the first throw out washing utilize streaming damping fluid resuspended, then add the Ligands being combined with fluorescence molecule, and centrifugal in conjunction with carrying out second time after 15-45 minute in 2-8 DEG C, obtain the second throw out;
3), by centrifugal after the second throw out washing, and flow cytometer detection is carried out after utilizing streaming damping fluid resuspended gained precipitation.
7. the method for detection CAR-T cell content according to claim 6, is characterized in that, in step 2) and step 3) in, described washing all adopts streaming damping fluid to carry out.
8. the method for the detection CAR-T cell content according to claim 6 or 7, is characterized in that, described first time, centrifugal centrifugal force was 500-1000g, and the time is 5-10min; The centrifugal centrifugal force of described second time is 500-1000g, and the time is 5-10min.
9. the capture probe described in any one of claim 1-4 is detecting the application in CAR-T cell content.
10. the application of the capture probe described in any one of claim 1-4 in the product for the preparation of detection CAR-T cell content.
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| CN201610079872.0A CN105504042A (en) | 2016-02-04 | 2016-02-04 | Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application |
| PCT/CN2016/096469 WO2017133222A1 (en) | 2016-02-04 | 2016-08-24 | Capture probe for detecting car-t cell, method for detecting cell content, and application |
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| CN201610079872.0A CN105504042A (en) | 2016-02-04 | 2016-02-04 | Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861733A (en) * | 2016-06-14 | 2016-08-17 | 宜明细胞生物科技有限公司 | Primer, probe and method for detecting CAR-T transduction efficiency |
| CN105950761A (en) * | 2016-06-24 | 2016-09-21 | 安徽未名细胞治疗有限公司 | Method for detecting number of CAR-T cells in bodies |
| CN106279419A (en) * | 2016-08-08 | 2017-01-04 | 吴江近岸蛋白质科技有限公司 | A kind of antibody binding proteins Protein L and preparation method thereof |
| CN108088783A (en) * | 2017-12-15 | 2018-05-29 | 湖南昭泰涌仁医疗创新有限公司 | The detection probe and its detection method of a kind of CAR-T cells and application |
| CN109856388A (en) * | 2018-11-29 | 2019-06-07 | 北京优迅医学检验实验室有限公司 | The catching method and capture kit of circulating tumor cell |
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| CN110894238A (en) * | 2019-11-25 | 2020-03-20 | 华道(上海)生物医药有限公司 | Monoclonal antibody for detecting CAR-T cells, kit and application |
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| WO2017143094A1 (en) | 2016-02-16 | 2017-08-24 | Dana-Farber Cancer Institute, Inc. | Immunotherapy compositions and methods |
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| CN105861733A (en) * | 2016-06-14 | 2016-08-17 | 宜明细胞生物科技有限公司 | Primer, probe and method for detecting CAR-T transduction efficiency |
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| CN109856388A (en) * | 2018-11-29 | 2019-06-07 | 北京优迅医学检验实验室有限公司 | The catching method and capture kit of circulating tumor cell |
| CN110894238A (en) * | 2019-11-25 | 2020-03-20 | 华道(上海)生物医药有限公司 | Monoclonal antibody for detecting CAR-T cells, kit and application |
| CN110894238B (en) * | 2019-11-25 | 2021-01-19 | 华道(上海)生物医药有限公司 | Monoclonal antibody for detecting CAR-T cells, kit and application |
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