WO2019129178A1 - Composition contenant un anticorps monoclonal anti-cd45 et méthode d'utilisation associée - Google Patents

Composition contenant un anticorps monoclonal anti-cd45 et méthode d'utilisation associée Download PDF

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WO2019129178A1
WO2019129178A1 PCT/CN2018/124695 CN2018124695W WO2019129178A1 WO 2019129178 A1 WO2019129178 A1 WO 2019129178A1 CN 2018124695 W CN2018124695 W CN 2018124695W WO 2019129178 A1 WO2019129178 A1 WO 2019129178A1
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mab
antibody
cells
blood
mabs
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PCT/CN2018/124695
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English (en)
Chinese (zh)
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钱其军
叶真龙
马硕
王欣玥
张晓霞
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上海白泽医学检验所有限公司
上海细胞治疗研究院
上海细胞治疗集团有限公司
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Publication of WO2019129178A1 publication Critical patent/WO2019129178A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the method of directly capturing by using CTCs surface markers is also called positive phase enrichment. This method is currently the most widely used positive phase enrichment method. The most typical representative is the Cellsearch system. Because CTCs belong to non-blood-derived epithelial cells, some CTCs express epithelial cell-specific molecules, Epithelial cell adhesion molecule (EpCAM), so this method mainly uses epithelial cell surface marker EpCAM antibody and even Linked to a carrier, such as a magnetic bead, to capture EpCAM-positive CTCs directly from the blood sample. However, the data suggest that not all solid tumors have EpCAM-positive expression, such as EpCAM, which is either low or negative on bladder cancer and malignant melanoma cells.
  • EpCAM Epithelial cell adhesion molecule
  • the tumor cells in the primary tumor tissue lose their polar epithelial cell characteristics (adhesive, flaky structure) and transform into interstitial cell characteristics with migration and invasion ability. (No cell polarity, loss of tight junctions between cells and cells).
  • these tumor cells undergo obvious cytoskeletal remodeling, which changes the expression of various EMT-related transcription factors and cell surface receptors. Therefore, the direct capture method using EpCAM is not high in specificity, and can not effectively capture CTCs of various tumors, and the capture efficiency and recovery rate are low.
  • cell surface EpCAM protein antigens are extremely active signaling-inducing factors, so the use of EpCAM antibody-coupled magnetic beads to capture CTCs is likely to trigger and activate a series of intracellular signaling pathways. Therefore, most of the CTCs captured by this method may not be CTCs in the natural state, and the captured CTCs have low cell activity, unable to carry out cell culture in the later stage, and are not conducive to cell separation, and are used in downstream technology. Research.
  • Density gradient centrifugation is used instead of red blood cell lysate to eliminate red blood cells, which greatly reduces the damage to CTCs, and retains the characteristics and characteristics of CTCs. Although this method has high sensitivity, it can effectively separate CTCs in the natural state, but the residual white blood cells are excessive.
  • SE-iFISH is a method in which red blood cells and plasma are removed by density gradient centrifugation, and immunomagnetic beads coupled with various leukocyte subclass antibodies are added to incubate to bind white blood cells, and magnetic force is used to remove white blood cells. effect. However, the final leukocyte remaining amount used to remove leukocytes is usually as high as 5x10 3 -1.5x10 4 /mL.
  • one of the main criteria for identifying CTCs is chromosome 8 polyploid, while macrophages in leukocytes are also polyploids of chromosome 8, and polyploid macrophages are likely to be present in such high leukocyte residuals.
  • the presence of this it will easily lead to the occurrence of false positive CTCs; and excessive white blood cell residue, when the microscope automatically scans and filters the white blood cells with CD45 positive expression, if there is CTC in the white blood cell agglomeration, it is easy to be automatically determined by the scanning system. A false negative thus filters out the cells.
  • the excessive number of residual white blood cells will bring greater difficulties and background effects to downstream CTCs cell sequencing and CTCs culture.
  • the MINDEC method is another negative enrichment method. Compared with the SE-iFISH method, the number of white blood cells remaining in the process using MINDEC is significantly less.
  • This method first uses density gradient centrifugation to remove red blood cells and plasma, and then uses a combination of antibodies against different leukocyte subclasses to capture white blood cells to enrich CTCs. In contrast to the SE-iFISH method, this method is first incubated with a biotin-labeled antibody in a mononuclear cell suspension after removal of red blood cells and plasma, followed by streptavidin. The labeled magnetic beads allow it to bind to the antibody.
  • the MINDEC method has more magnetic bead removal times than SE-iFISH, which has a better effect of removing magnetic beads and white blood cells, and at the same time avoids the risk of CTCs being removed by magnetic beads. Recovery rate of CTCs.
  • the application of MINDEC method has higher leukocyte removal efficiency, and the final number of remaining leukocyte cells is 437 ⁇ 350/mL, which effectively reduces the possibility of false positive and false negative detection. However, because of the different combinations of antibodies against the leukocyte subclass, the amount of antibody required is higher and the cost is higher.
  • the antibody used by MINDEC is a murine monoclonal antibody, and the antibody extracted from the mouse has low affinity for human leukocytes, so the demand for the mouse monoclonal antibody is large in the MINDEC method.
  • a first aspect of the invention provides a composition comprising an antibody comprising two or more anti-CD45 rabbit monoclonal antibodies.
  • the antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are each different.
  • the anti-CD45 mAb is a rabbit anti-human monoclonal antibody.
  • the anti-CD45 mAb has an affinity coefficient ⁇ 1.0 x 10 -10 M.
  • the affinity of at least one of the anti-CD45 mAbs is ⁇ 4.0 x 10 -11 M.
  • the antibody binds to an affinity tag; the affinity tag is preferably biotin.
  • the mass ratio of the highest affinity anti-CD45 rabbit monoclonal antibody to the total content of all other anti-CD45 rabbit monoclonal antibodies in the anti-CD45 mAb of the composition is 1-10: 1; preferably 5:1.
  • the antibody-containing composition is used with anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb for enrichment of circulating tumor cells.
  • the antibody-containing composition comprises an anti-CD16 mAb, an anti-CD19 mAb, an anti-CD235a mAb, and two or more anti-CD45 mAbs, wherein the two or The antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are different; preferably, the mAbs are all coupled with an affinity tag; preferably, the affinity tag is a biotin molecule.
  • the two or more anti-CD45 mAbs have an affinity coefficient of less than 1.0 x 10 -10 M; preferably, the two or more anti-CD45 mAbs At least one of the affinity coefficients is less than 4.0 x 10 -11 M.
  • the species source of the monoclonal antibodies in the composition is the same or a different species source; preferably, the species source of one of the two or more anti-CD45 mAbs is The source of the rabbit, other anti-CD45 mAb species is a murine source; more preferably, the species source of the two or more anti-CD45 mAbs is a rabbit source.
  • the anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb are each independently a rabbit anti-human monoclonal antibody or a murine anti-human monoclonal antibody.
  • the two or more anti-CD45 mAbs are rabbit anti-human CD45 monoclonal antibodies
  • the anti-CD16 mAb is a murine anti-human CD16 monoclonal antibody
  • the anti-CD19 The monoclonal antibody is a murine anti-human CD19 monoclonal antibody
  • the anti-CD235a monoclonal antibody is a murine anti-human CD235a monoclonal antibody.
  • the invention provides a kit comprising the antibody-containing composition described herein.
  • the kit further comprises magnetic beads coupled to a ligand capable of binding to the affinity tag of the preceding embodiments; preferably, the ligand is Streptavidin.
  • the antibody-containing composition and magnetic beads are each placed in separate containers.
  • the invention provides a combination of two or more CD45 monoclonal antibodies in combination with anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in leukocytes isolated from blood or in enriched peripheral blood circulating tumor cells Applications, or in the preparation of reagents or kits for isolating leukocytes in blood or for enriching peripheral blood circulating tumor cells.
  • the anti-CD45 mAb composition comprises two or more anti-CD45 mAbs that target different antigenic determinants of the CD45 molecule, one of which is a rabbit source, the other one For the mouse source.
  • the ratio of the highest affinity anti-CD45 rabbit monoclonal antibody in the anti-CD45 mAb composition to the sum of the masses of the other anti-CD45 rabbit monoclonal antibodies is 1-10:1; preferably 5 :1.
  • the present invention also provides a method of separating leukocytes in blood, the method comprising the step of contacting blood with a composition described herein.
  • the blood is blood from which red blood cells and plasma are removed.
  • the present invention provides a method of enriching circulating tumor cells, the method comprising:
  • the step (4) comprises mixing the liquid obtained in the step (3) with the antibody-containing composition, incubating for 10 to 30 minutes, and then centrifuging.
  • the step (5) comprises adding streptavidin-coupled magnetic beads for 10 to 20 minutes.
  • the step (6) comprises, after the end of the incubation, placing the container containing the liquid and the magnetic beads on a magnetic stand, allowing to stand, so that the magnetic beads are adsorbed on the magnet, and the suction is not performed.
  • a liquid containing a magnetic bead portion is not performed.
  • Figure 1 Total number of residual white blood cells after treatment with high affinity and low affinity CD45 antibody at a mass ratio of 1:1, 5:1, 10:1.
  • Figure 2 Total number of CTCs detected after treatment with high affinity and low affinity CD45 antibody at a mass ratio of 1:1, 5:1, 10:1.
  • Figure 3 Detection rate of CTCs by the method of the invention and the control method.
  • Figure 5 Total number of suspected cells during the identification of CTCs by the method of the invention and the control method.
  • PT.1 (Patient 1) in Figure 1-5 represents Patient 1, and so on.
  • the present invention aims to solve the problem that the circulating tumor cell recovery rate is not high, the white blood cell identification is incomplete, and the active circulating tumor cells cannot be accurately obtained due to excessive residual leukocyte cells in the existing circulating tumor cell isolation method.
  • an antibody-containing composition also referred to as "antibody composition”
  • the antibody comprising the antibody comprising two or more anti-CD45
  • Monoclonal antibodies in combination with anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb, are used for enrichment and identification of CTCs.
  • Anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb suitable for use herein can be a variety of anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb, which are well known in the art.
  • the present invention can be practiced using commercially available anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb, as can be obtained from Abcam, Thermo Fisher.
  • these monoclonal antibodies can be prepared by themselves using techniques well known in the art, such as hybridoma technology.
  • two or more anti-CD45 mAbs are rabbit anti-human CD45 mAb.
  • the affinity coefficient of the two or more anti-CD45 mAbs is ⁇ 1.0 x 10 -10 M. More preferably, the affinity coefficient of at least one of the two or more anti-CD45 mAbs is ⁇ 4.0 ⁇ 10 -11 M, preferably ⁇ 2.0 ⁇ 10 -11 M, more preferably ⁇ 1.0 ⁇ 10 -11 M.
  • the affinity coefficients of the two or more anti-CD45 mAbs are ⁇ 3.0 x 10 -11 M.
  • the antigenic determinants on the CD45 antigen molecule recognized by the two or more anti-CD45 mAbs are different.
  • the two or more anti-CD45 mAbs are rabbit anti-human CD45 mAbs with different affinities, preferably having an affinity coefficient of ⁇ 4.0 x 10 -11 M.
  • the ratio of the mass of the most affinity-resistant anti-CD45 mAb to the sum of the masses of the other anti-CD45 mAbs in the composition may range from 1 to 10:1, preferably from 3 to 7:1. In certain embodiments, the ratio is 5:1.
  • the amount of the antibody can be measured in micrograms.
  • the anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb may be rabbit anti-human mAb or murine anti-human mAb.
  • the affinity coefficient of rabbit anti-human monoclonal antibody suitable for use herein is ⁇ 4.0 ⁇ 10 -11 M, preferably ⁇ 2.0 ⁇ 10 -11 M, more preferably ⁇ 1.0 ⁇ 10 -11 M.
  • the antibody in the antibody-containing compositions described herein, may be two or more rabbit anti-human CD45 mAb, murine anti-human CD16 mAb, murine anti-human CD19 mAb, and mouse anti- Human CD235a monoclonal antibody.
  • the mass ratio of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb and anti-CD235a mAb in the composition may range from 1:1 to 5:1 to 5:1 to 5, It is preferably in the range of 1:2 to 4:2 to 4:2 to 4.
  • the mass ratio of two or more anti-CD45 mAb, anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb is 1:3:3:3.
  • the antibody in the antibody-containing compositions described herein, is two or more rabbit anti-human CD45 mAb, murine anti-human CD16 mAb, murine anti-human CD19 mAb, and murine anti-human
  • the CD235a monoclonal antibody has a mass ratio in the range of 1:1 to 5:1 to 5:1 to 5, preferably in the range of 1:2 to 4:2 to 4:2 to 4, more preferably 1:3. :3:3.
  • Suitable solvents may be included in the compositions including, but not limited to, PBS, EDTA, and BSA.
  • kits comprising a composition described herein.
  • the kit may also contain magnetic beads.
  • the composition and magnetic beads are dispensed in separate containers.
  • the magnetic beads can be various magnetic beads known in the art for cell separation, including magnetic beads of any suitable size and material.
  • streptavidin or other suitable molecule is coupled to the magnetic beads to bind to an antibody that is coupled to biotin or a corresponding molecule to separate leukocytes captured by the antibody molecules.
  • the antibody compositions herein can be used to separate leukocytes in blood or enrich for circulating tumor cells.
  • a method of isolating leukocytes in blood comprising the step of contacting an antibody composition described herein with the blood.
  • the contacting can be carried out according to the technical means conventional in the art, and then the magnetic beads coupled with the corresponding binding molecules are added to adsorb the leukocytes captured by the antibody molecules onto the magnetic beads, and the blood beads are separated from the blood by separating them.
  • White blood cells are isolated.
  • blood is usually referred to as human peripheral blood unless otherwise stated.
  • plasma and red blood cells in the blood can be removed using conventional techniques in the art. For example, plasma can be removed by conventional centrifugation and red blood cells can be removed by density gradient centrifugation.
  • a method of enriching circulating tumor cells comprising:
  • the magnetic beads are removed to enrich the circulating tumor cells.
  • the blood When serum is removed, the blood can be mixed and centrifuged, and the supernatant is washed away.
  • the precipitate obtained after plasma removal can be mixed with a washing buffer and a density gradient separation solution for density gradient centrifugation.
  • the mixture After centrifugation, the mixture is usually layered in three layers, the red precipitated layer being red blood cells and the middle being a white film layer. Usually, the middle white film layer is removed first, and then the remaining liquid is taken up. The liquid is washed several times and centrifuged to remove impurities such as proteins and platelets in the solution. These steps can be accomplished using techniques conventional in the art. For example, the time, the rotation speed, and the like of the centrifugation can be carried out under normal conditions.
  • the centrifugation conditions for removing plasma may be 150 to 300 g for 10 to 20 minutes; the centrifugation conditions for removing red blood cells may be 300 to 500 g for 20 to 30 minutes; and the centrifugation conditions for removing impurities such as proteins and platelets may be 500 to 700 g, 3 ⁇ 8 minutes.
  • the antibody composition of the present invention is added, and after incubation for a period of time, the magnetic beads coupled with the corresponding molecules are added, and then incubated for a period of time, and the magnetic beads bound to the white blood cells are separated by magnetic force, thereby enriching the circulating tumor cells.
  • the amount of the antibody composition to be added and the amount of the magnetic beads to be added can be determined according to actual conditions.
  • the incubation time after mixing with the antibody composition is usually in the range of 10 to 30 minutes, and the incubation temperature may be in the range of 2 to 8 °C.
  • the separation washing solution may be added, mixed, and centrifuged to remove the antibody that does not bind the blood cells. It can be centrifuged for 5 to 15 minutes under conditions of 200 to 400 g.
  • the incubation time with the magnetic beads is usually in the range of 10 to 20 minutes, and the incubation temperature is usually 2 to 8 °C.
  • the cells can be resuspended by separating the cleaning solution, and gently mixed with a gun head, then placed on a magnetic stand for magnetic separation, and the supernatant is aspirated.
  • the supernatant can be centrifuged at 2 to 8 ° C, and the centrifugation conditions can be 200 to 400 g for 5 to 15 minutes. It should be understood that the entire process of isolating white blood cells is usually carried out at a temperature of 2 to 8 °C.
  • the present invention is based on two or more high affinity anti-CD45 mAbs (the affinity for human leukocytes is much higher than the commonly used murine monoclonal antibody, up to 3.6 ⁇ 10 -11 M), when When the single cell suspension is incubated, it can bind to leukocytes more efficiently, so the anti-CD45 monoclonal antibody required by the method of the present invention can be reduced by at least half compared to the amount of murine monoclonal antibody of MINDEC. After that, the white blood cells in the suspension can be removed more effectively by combining the magnetic beads. Under the same antibody dosage, the method of the invention can control the remaining white blood cell residual amount to 200 ⁇ 50/6.0mL, further increasing the recovery rate of CTCs. Reduce the appearance of suspected cells and reduce the difficulty of identifying CTCs.
  • the method of the invention can better label various leukocyte subclasses due to the high affinity of the antibody combination, thereby better removing white blood cells and enriching CTCs more efficiently. It does not damage CTC cells, so that CTC cells maintain a good natural state and cell morphology, and can perform genomic, transcriptome and proteomic analysis on subsequent single (multiple) circulating tumor cells or culture of circulating tumor cells after enrichment. .
  • the recovery rate of the circulating tumor cells of the present invention is as high as 95% or more; the present invention also has the advantage of high sensitivity, and can stably detect circulating tumor cells in samples with extremely small blood volume.
  • combinations of two or more of the high affinity anti-CD45 mAbs described herein are also provided herein for isolating leukocytes in the blood together with anti-CD16 mAb, anti-CD19 mAb, and anti-CD235a mAb or enrichment Use in peripheral blood circulating tumor cells, or in the preparation of reagents or kits for isolating leukocytes in blood or for enriching peripheral blood circulating tumor cells.
  • CTCs circulating tumor cells
  • CD45 common antigen on the surface of leukocytes
  • CD16 NK cells and neutrophil antigens
  • CD19 B cell antigen
  • CD235a blood glycoprotein a.
  • Magnetic beads Dynabeads MyOne 1 ⁇ m, streptavidin coated, Thermo Fisher.
  • DAPI 4',6-diamidino-2-phenylindole
  • Step 1 Aspirate the corresponding amount of the magnetic bead mixture from the magnetic bead mixture labeled with streptavidin into a 2 mL EP tube, and place the EP tube on the magnetic stand for 3 minutes, the configuration of the magnetic beads.
  • the amount is 100 ⁇ L per 1 ⁇ 10 7 cells;
  • Step 2 After the magnetic beads are adsorbed on the magnetic frame, use a vacuum pump to aspirate the magnetic bead preservation solution, add at least 1 mL of the separated cleaning solution, mix and mix slowly, place on a magnetic stand for 3 minutes, and use a vacuum pump to aspirate the cleaning solution. ;
  • Step 3 Add an equal volume of separation cleaning solution to the magnetic beads mixed with the first extraction, mix slowly, and let stand at room temperature for subsequent operations.
  • Step 1 Collect 6.0 mL of peripheral blood, mix the gently-tailed tail of the blood collection tube and centrifuge, and centrifuge for 200 g for 15 minutes; the supernatant (plasma) after centrifugation is vacuumed with a vacuum pump;
  • Step 2 Add an equal volume of cell cleaning solution to the blood cells precipitated in the blood collection tube, and gently mix the head and tail;
  • Step 3 Add 3 mL of density gradient separation solution Ficoll-Pague (1.086 g/mL) to the labeled 15 mL centrifuge tube A;
  • Step 4 slowly add the above blood cell mixture to the upper part of the separation liquid in the centrifuge tube A, and centrifuge in a centrifuge, and centrifuge at 400 g for 20-30 minutes;
  • Step 6 Add 5 times the cell volume of the above liquid volume to the centrifuge tube B, centrifuge the cells for washing, centrifuge at 600 g for 5 minutes, discard the supernatant to 100 ⁇ L, and mix and resuspend the cells by pipetting.
  • Step 1 100 ⁇ L of the cell suspension obtained in the above procedure was transferred to a new 15 mL centrifuge tube C, and a biotin-labeled high-affinity antibody mixture (rabbit anti-human CD45 mAb, mouse anti-human CD16 mAb, mouse anti-mouse) was added.
  • Step 2 Mix gently and mix, incubate at 4 ° C for 20 minutes, mix once every 10 minutes;
  • Step 3 After the end of the incubation, the separation washing solution is added to remove the unbound blood cells, the head and tail are mixed upside down several times, and the supernatant is centrifuged to 100 ⁇ L, and the centrifugation condition is 300 g for 10 minutes (2 ° C - 8 ° C);
  • Step 4 Add 3 mL of separation washing solution and mix and resuspend the cells;
  • Step 6 After the end of the incubation, add 3 mL of the separated washing solution to the centrifuge tube C; resuspend the magnetic beads-bound cells, mix gently with a pipette tip, and remember not to generate air bubbles; then place on a magnetic stand for 3 minutes;
  • Step 7 Carefully avoid the magnetic beads transfer supernatant to a new 15 mL centrifuge tube D;
  • Step 9 Carefully avoid the magnetic beads transfer supernatant to the 15mL centrifuge tube D;
  • Step 11 Transfer all the liquid from the centrifuge tube D to the labeled 15 mL centrifuge tube E; discard the supernatant to 50 ⁇ L and centrifuge at 300 g for 10 minutes (4 ° C).
  • Step 2 Add 1 ⁇ L of each fluorescent antibody (CD45-AF594, tumor-labeled fluorescent antibody), incubate at room temperature for 20 minutes in the dark, and gently mix once every 10 minutes using an oscillating mixer;
  • each fluorescent antibody CD45-AF594, tumor-labeled fluorescent antibody
  • Step 3 Add the cell washing solution to 14 mL, trim, mix by inversion, centrifuge at room temperature, centrifuge at 950 g for 4 minutes, discard the supernatant to 100 ⁇ L;
  • Step 4 Add 100 ⁇ L of 4% paraformaldehyde to the above 100 ⁇ L of cell suspension, and gently mix and mix using a pipette tip;
  • Step 8 gently add 200 ⁇ L of 1 ⁇ PBS along the inner corner of the specimen frame, and immediately discard it for 2 times;
  • Step 9 gently add 200 ⁇ L of 1 ⁇ PBS along the inner corner of the specimen frame, let stand for 2 minutes, and discard it for a total of two times;
  • Step 10 gently add 200 ⁇ L of absolute ethanol along the inner corner of the specimen frame, and immediately discard it, repeating 2 times;
  • Step 11 Insert the slide into the dyeing tank containing absolute ethanol for 2 minutes, remove the slide, erect on the filter paper, completely absorb the residual liquid from the slide, and gently slide the slide to the surface completely using a micro-hair dryer. Rear;
  • Step 12 The following operations should be protected from light. Immediately after drying the slide, 10 ⁇ L of the chromosome 18 FISH probe was added to the center of the specimen frame, and then the cover slip was placed on the probe solution using forceps to make the liquid. Spread to the entire specimen frame; if necessary, for example, there are air bubbles in the specimen frame, use a forceps to gently press the cover slip so that the probe fluid covers the entire specimen frame and discharge the air bubbles;
  • Step 13 Mounting: Cut off the tip of the 1 mL sampler, and seal each side of the coverslip with 250 ⁇ L of sealant after each slide, and put it directly into the hybridization instrument;
  • Step 14 Hybridization: denaturation at 76 ° C for 10 minutes; hybridization at 37 ° C for 4 hours;
  • Step 15 After the hybridization is completed, remove the slide; gently press the corner of the cover glass by hand, use a pair of tweezers to remove the sealant, place the slide on the dyeing tank preheated to room temperature, and let stand for 1 minute, then gently shake the dyeing tank. Until the coverslip falls off. After the coverslip is detached, the slide is continuously left in the cylinder for 1 minute at room temperature, and then the residual liquid flowing down the slide is dried by using a filter paper, and the liquid outside the specimen frame is wiped off;
  • Step 16 After gently dropping 200 ⁇ L of 2x SSC/0.1% NP-40 along the inner corner of the specimen frame, immediately aspirate the solution and repeat 2 times;
  • Step 18 Take 10 ⁇ L of fluorescent preservation droplets and add them to the center of the specimen frame. After placing the coverslips, fix the coverslips in one hand to prevent them from sliding. The other hand gently squeezes and absorbs the excess liquid overflowing with vacuum pump tips or filter paper. Sealed specimens;
  • the blood of the three patients was divided into 3 tubes, 2 ml each, and the total number of residual white blood cells and CTC detected by the high-affinity and low-affinity CD45 antibody mass ratios of 1:1, 5:1, and 10:1, respectively. Compare.
  • the total number of CTCs detected for each sample was counted and the results are shown in Fig. 2.
  • the results show that the mass ratio of 5:1 is better than 1:1 and 10:1.
  • the possible reason is that the amount of high-affinity antibodies in the 1:1 dosage is too low, and the white blood cells remain too much, causing the CTC cells to be missed; and in the ratio of 10:1, the amount of high-affinity antibodies is too high, so that the CTC cells It is removed by leukocyte agglomeration, resulting in missed detection.
  • the total number of cells per sample was scanned and the results are shown in FIG.
  • the results show that the total amount of residual blood-derived white blood cells of the present invention is lower than that of the control, indicating that the present invention has the ability to efficiently enrich CTCs.

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Abstract

La présente invention concerne une méthode d'enrichissement et d'identification de cellules tumorales circulantes (CTC) en fonction d'au moins deux anticorps monoclonaux de lapin anti-CD45 à haute affinité, ainsi qu'une application associée. Plus précisément, la présente invention concerne une composition contenant au moins deux anticorps monoclonaux de lapin CD45, les anticorps pouvant reconnaître différents déterminants antigéniques sur des molécules d'antigène CD45. La présente invention concerne également une méthode de séparation de leucocytes et d'enrichissement et d'identification de CTC à l'aide de ladite composition conjointement avec un anticorps monoclonal anti-CD16, un anticorps monoclonal anti-CD19 et un anticorps monoclonal anti-CD235a. La combinaison d'anticorps dans la présente invention implique des anticorps monoclonaux de lapin présentant une affinité et une spécificité élevées, permet de mieux marquer diverses sous-classes de leucocytes, ce qui permet de mieux éliminer les leucocytes et d'enrichir et d'identifier plus efficacement les CTC, et de ne pas endommager les cellules CTC, de telle sorte que les cellules CTC puissent conserver un bon état naturel et une bonne forme de cellule.
PCT/CN2018/124695 2017-12-29 2018-12-28 Composition contenant un anticorps monoclonal anti-cd45 et méthode d'utilisation associée WO2019129178A1 (fr)

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