Magnetic bead for T cell extraction and preparation method and application thereof

Abstract

The invention provides a magnetic bead for T cell extraction and a preparation method and application thereof. Comprising the following components: A-L-B, A is an epoxy magnetic bead nanoparticle, B is a sulfhydrylated T cell antibody, L is a linker molecule, and L is covalently linked with an epoxy group of A and a sulfydryl group of B respectively. The magnetic bead provided by the invention has the advantages of high dissolution, stable dispersion and capability of screening and separating T cells.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular biology, and in particular to a magnetic bead for T cell extraction, its preparation method, and its application. Background Technology

[0002] CAR-T therapy, also known as chimeric antigen receptor T-cell therapy, is a novel immune cell-based precise targeted therapy technology for tumors in clinical practice.

[0003] Through genetic engineering, the patient's T cells are first activated, then a tumor chimeric antigen receptor is added to the activated T cells. Finally, with the help of specialized instruments or equipment, CAR-T cells are obtained and then reinfused into the patient. These modified CAR-T cells will attack and destroy tumor cells, ultimately achieving the goal of treating tumor diseases.

[0004] In the process of constructing and expanding CAR-T cells in vitro, the activation and proliferation of T cells require the presence of co-stimulatory molecules, which also contribute to the production of CAR-T cell cytokines. In vitro, this process is typically achieved using magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies.

[0005] Although CAR-T therapy has become a promising treatment for cancer, there are still areas for improvement. Currently, the preparation of CAR-T magnetic beads is controlled by a few foreign manufacturers, who essentially monopolize the preparation process for all cell sorting magnetic beads, from small-scale to large-scale sorting, and from high-frequency cells to rare subpopulations. Domestic patients wishing to use CAR-T therapy must rely on imported magnetic beads, which is prohibitively expensive and unaffordable for ordinary families. Furthermore, imported magnetic beads require extremely high equipment compatibility; they can only be used with complete solutions developed by the manufacturers, including cell sorting magnetic beads, tubing, buffer solutions, and cell culture products. If any equipment in the solution is not original equipment, subsequent treatment steps cannot be performed. Especially regarding the accuracy of cell screening, this needs to be examined in long-term clinical studies. Therefore, exploring a universally applicable method for the preparation and application of magnetic beads capable of efficiently capturing lymphocytes (T cells) is currently a key research focus and challenge. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a magnetic bead for T cell extraction, its preparation method, and its application, resulting in a universally applicable magnetic bead capable of efficiently capturing lymphocytes (T cells).

[0007] In a first aspect, the present invention provides magnetic beads for T cell extraction, comprising components of the following formula:

[0008] ALB, where A is an epoxy-based magnetic bead nanoparticle, B is a thiolized T-cell antibody, and L is a linker molecule, which is covalently linked to the epoxy group of A and the thiol group of B, respectively.

[0009] In one embodiment of the present invention, the epoxy-based magnetic bead nanoparticles are based on a magnetic metal oxide core, the surface of which is coated with polysaccharides and epoxy groups are modified on the surface of the polysaccharides.

[0010] In one embodiment of the present invention, the thiolized T-cell antibody has a T-cell antibody as its core and thiol groups are modified on the surface of the T-cell antibody.

[0011] In one embodiment of the present invention, the T cell antibody includes CD4 antibody and CD8 antibody.

[0012] In one embodiment of the present invention, the linking molecule includes a biocompatible polymer with active groups and complexing groups.

[0013] In one embodiment of the present invention, the biocompatible polymer includes linear, branched polyethylene glycol (PEG), and also includes one of the block copolymers formed by linear, branched polyethylene glycol and polyacrylic acid (PAA), polymethacrylic acid (PMA), polyethyleneimine (PEI), polyalanine, polylysine, polyleucine, polyglutamic acid, polyaspartic acid, or polylactic acid (PLA).

[0014] In one embodiment of the present invention, the active group is covalently linked to the thiol group of the T-cell antibody; the complexing group is covalently linked to the epoxy group of the magnetic bead nanoparticles.

[0015] In one embodiment of the present invention, the active group includes maleamide subunit, alkynyl, alkenyl, azide, cyano, enolyl, or enol ether. Maleamide subunit is preferred.

[0016] In one embodiment of the present invention, the complexing group includes a carboxyl group, an amino group, a hydroxyl group, a thiol group, or a phosphate group. Preferably, it is an amino group.

[0017] In one specific embodiment of the present invention, the biocompatible polymers include NHS-PEG-MAL(2k) and NHS-PEG(550).

[0018] In one embodiment of the present invention, the ratio of the linking molecule: epoxy-based magnetic bead nanoparticles: thiolized T-cell antibody by mass is 25-4:1:10-2; preferably 15-7:1:6-3.

[0019] In a second aspect, the present invention provides a method for preparing magnetic beads for T cell extraction, comprising covalently linking epoxy-based magnetic bead nanoparticles and thiolized T cell antibodies using linker molecules.

[0020] In one embodiment of the present invention, the preparation method of the epoxy-based magnetic bead nanoparticles includes: 1) dissolving a magnetic metal compound and a polysaccharide in water and reacting them under an inert gas to obtain a polysaccharide-coated magnetic metal oxide solution; 2) adding an alkaline solution and an epoxy-based molecule to step 1) to react and obtain epoxy-based magnetic bead nanoparticles.

[0021] In one embodiment of the present invention, the magnetic metal compound includes hydrated and anhydrous chloride, nitrate, and / or sulfate salts of magnetic metal elements. The paramagnetic metal elements include, but are not limited to, iron, gadolinium, neodymium, promethium, samarium, europium, terbium, dysprosium, holmium, erbium, and thulium. Iron is preferred.

[0022] In one specific embodiment of the present invention, the magnetic metal compound includes ferric chloride and ferrous chloride. The magnetic metal oxide includes iron(III) oxide and ferric oxide.

[0023] In one embodiment of the present invention, the polysaccharide includes glucan.

[0024] In one specific embodiment of the present invention, the molar ratio of the magnetic metal compound to the polysaccharide is 10:1 to 100:1, preferably 20:1 to 75:1.

[0025] In one specific embodiment of the present invention, the concentration of the magnetic metal compound is 0.001 mol / L to 0.2 mol / L, preferably 0.01 to 0.1 mol / L.

[0026] In a specific embodiment of the present invention, in step 1), the reaction temperature is controlled at 25-40°C, preferably 25-35°C, under an inert gas atmosphere; the reaction time is 0.2-5 hours, preferably 0.5-2.5 hours.

[0027] In one embodiment of the present invention, the alkaline solution comprises sodium hydroxide, potassium hydroxide, calcium hydroxide, and ammonia. The volume of the alkaline solution added is 0.01 to 0.2 times that of the polysaccharide-coated magnetic metal oxide solution.

[0028] In one embodiment of the present invention, the molecule with an epoxy group includes epichlorohydrin.

[0029] In one specific embodiment of the present invention, the concentration of the molecule with epoxy group is 0.003 mol / L to 0.8 mol / L, preferably 0.03 to 0.6 mol / L.

[0030] In one embodiment of the present invention, the method for preparing the thiolated T-cell antibody includes: reacting a thiolation reagent with a T-cell antibody to obtain a thiolated T-cell antibody.

[0031] In one embodiment of the present invention, the thioacetic reagent includes thioacetic acid, hydrogen sulfide, thiolates (such as sodium thiocyanate, potassium thiocyanate, or TCEP), acetylthiol, or benzylmercaptoacetate. In one embodiment of the present invention, the reaction time is 15-45 min.

[0032] In one embodiment of the present invention, the mass ratio of the thiolized T-cell antibody to the epoxy magnetic bead nanoparticles is 3:1-7:1.

[0033] In one embodiment of the present invention, the method for preparing magnetic beads for T cell extraction includes the following steps: S1 covalently linking a linker molecule and epoxy-based magnetic bead nanoparticles; S2 adding a thiolized T cell antibody in step S1 to react and obtain the magnetic beads.

[0034] In one embodiment of the present invention, in step S1, the covalent connection time is 1 to 100 hours, preferably 2 to 40 hours.

[0035] In one embodiment of the present invention, the reaction time in step S2 is 20-60 min.

[0036] In one specific embodiment of the present invention, a method for preparing magnetic beads for T cell extraction includes the following steps:

[0037] (1) In a reaction vessel, an inorganic metal compound (such as ferric chloride, ferrous chloride, etc.) is dissolved in an aqueous phase to form a mixed reaction solution, wherein the concentration of the inorganic metal compound in the reaction solution is 0.001 mol / L to 0.2 mol / L, preferably 0.01 to 0.1 mol / L;

[0038] (2) Inert gas is introduced to remove oxygen from the reaction system, and the reaction liquid from step (1) is stirred at room temperature to obtain nanoparticles with narrow particle size distribution and controllable particle size; the reaction temperature is controlled at 25-40℃, preferably 25-35℃; the reaction time is 0.2-5 hours, preferably 0.5-2.5 hours.

[0039] (3) Add an alkaline solution (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) with a volume of 0.01 to 0.2 times the volume of the reaction solution to the reaction solution in step (2), and add a certain amount of epichlorohydrin. Finally, adjust the pH with an acidic solution (hydrochloric acid, acetic acid, sulfuric acid, etc.) to obtain the precursor of magnetic bead nanoparticles.

[0040] (4) The magnetic bead nanoparticle solution obtained in step (3) is reacted with a biocompatible polymer dissolved in the aqueous phase for 1 to 100 hours, preferably 2 to 40 hours; the reaction yields magnetic bead nanoparticles without grafted lymphocyte T cell antibodies.

[0041] (5) Add sulfhydrylating reagent to the magnetic bead nanoparticle solution obtained in step (4), and then add sulfhydrylated antibody to the magnetic bead nanoparticle solution to prepare magnetic bead nanoparticles with screening and separation capabilities.

[0042] In a third aspect, the present invention provides the use of the above-described magnetic beads in the preparation of one or more of the following application reagents:

[0043] 1) T cell extraction, separation and screening; 2) In vivo tracing of lymphocytes; 3) Evaluation and monitoring of the efficacy of CAR-T therapy; 4) Early monitoring of tumor metastasis.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1) This invention utilizes a complexation reaction to crosslink inorganic metal compounds with inorganic substances in an aqueous phase at room temperature, preparing a biocompatible magnetic bead nanoparticle precursor with high crystallinity, narrow particle size distribution, and controllable particle size, possessing surface-active functional groups. Thiol groups are introduced into antibodies via ether bonding, and the antibodies are grafted onto the surface of the magnetic beads through click chemistry reactions between the thiol groups and functionalized groups. This produces magnetic bead nanoparticles that are highly soluble and stably dispersed in physiological buffer solutions and possess screening and separation capabilities. The screened and separated magnetic bead nanoparticle powder samples remain completely soluble in water and physiological buffer solutions after long-term storage, forming stable colloidal solutions. The magnetic bead nanoparticles used for screening and separation exhibit high stability in physiological buffer solutions.

[0046] 2) The magnetic beads of the present invention are paramagnetic, with a particle size of 1-200 nm and a relative standard deviation of less than 10% in particle size. By adjusting the ratio of iron oxide to polysaccharide, nanoparticles of different particle sizes can be obtained.

[0047] 3) This invention improves T cell capture efficiency by adjusting the ratio of iron oxide to polysaccharides and the ratio of antibodies to magnetic bead nanoparticles. The efficiency is significantly higher than that of clinically used magnetic beads when the antibody type is the same. In summary, magnetic beads exhibit excellent T cell capture efficiency.

[0048] 4) The magnetic beads of this invention achieve highly efficient and specific recognition of lymphocyte-associated antigens, enabling efficient screening and separation. This effectively improves the current situation where magnetic beads are largely dependent on imports, promoting the development and progress of CAR-T therapy. By preparing specific screening magnetic beads and utilizing the properties of paramagnetic metal oxides, an in vivo tracking technology for lymphocyte-associated antigens can be established, enabling the evaluation and monitoring of treatment effects. By detecting the status of magnetic beads in circulating tumor cells, it is possible to provide early warning of whether malignant tumors have metastasized, and the recurrence of malignant tumors can be inhibited based on the detection results. Therefore, these magnetic beads have significant application prospects and research value in the diagnosis and treatment of malignant tumors. Attached Figure Description

[0049] Figure 1 This is the synthesis scheme for screening magnetic beads in this invention;

[0050] Figure 2 This is a TEM image of the magnetic beads being screened in Embodiment 1 of the present invention;

[0051] Figure 3 The graphs show the particle size distribution and potential comparison before and after cross-linking and cross-linking PEG of the magnetic beads in Example 1 of this invention.

[0052] Figure 4 This is a TEM image of the magnetic beads being screened in Embodiment 2 of the present invention;

[0053] Figure 5 The image shows a line graph of particle size distribution and a potential comparison graph before and after cross-linking and cross-linking PEG of the magnetic beads in Example 2 of this invention.

[0054] Figure 6 This is a TEM image of the magnetic beads being screened in Embodiment 3 of the present invention;

[0055] Figure 7 The image shows a line graph of particle size distribution and a potential comparison graph before and after cross-linking and cross-linking PEG of the magnetic beads in Example 3 of this invention.

[0056] Figure 8 This invention is used to screen the cytotoxicity of magnetic beads.

[0057] Figure 9 This is a flow cytometry result of the magnetic beads in Example 1 of this invention;

[0058] Figure 10 This is a flow cytometry result of the magnetic beads in Example 4 of this invention;

[0059] Figure 11 This is a flow cytometry result of the magnetic beads in Example 5 of this invention;

[0060] Figure 12 This is an in vivo tracer image showing the results of screening magnetic beads in this invention;

[0061] Figure 13 The image shows the flow cytometry results of the magnetic beads in Example 1 of this invention and commercial magnetic beads. Detailed Implementation

[0062] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0063] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0064] Example 1: Preparation of magnetic beads

[0065] 1. Using a pipette, measure 5 ml (0.06 mmol) of 12 mM FeCl3·6H2O solution, weigh 0.7 g FeCl2·4H2O (3.5 mmol) and 5 g (0.125 mmol) of dextran using an analytical balance, and add the samples to a round-bottom flask. The final volume of the reaction system is 110 ml. Inert gas is bubbled into the round-bottom flask for protection, and then 5 ml of ammonia water is quickly added. The mixture is stirred for 0.5 h. Inert gas protection is required throughout the entire reaction process. After the reaction is complete, centrifuge the reaction solution (3500 r / min, 10 min) to discard the precipitate. Transfer the supernatant to an ultrafiltration tube (molecular weight cutoff 30 kJ) and ultrafilter (4000 r / min, 30 min) three times. Collect the ultrafiltered solution, determine the iron concentration, and store it in the upper part of the refrigerator for later use.

[0066] 2. In a round-bottom flask, add the Fe3O4@DEX prepared in step 1, add 5 ml of sodium hydroxide solution with pH=10, and add 0.45 ml of epichlorohydrin (0.046 mol / L) dropwise. React at 50°C for 3 h. After the reaction is complete, add 0.65 ml of ammonia water dropwise to the flask and continue the reaction at 50°C for 3 h. After the reaction is complete, adjust the pH of the reaction system to neutral using 2% hydrochloric acid, ultrafilter, and determine the iron concentration for later use.

[0067] 3. Take 2 ml of the sample prepared in step 2, add 112 mg of NHS-PEG-MAL(2k) and react for 2 h. After the reaction is complete, add 30.932 mg of NHS-PEG(550) and react for 2 h. After the reaction is complete, ultrafilter and measure the iron concentration for later use.

[0068] 4. Take 0.1 mg of CD4 antibody, add 3.35 μg of thiolation reagent, and vortex for 2 hours. Then, quickly add the thiolation antibody to a centrifuge tube containing 12.5 μg of magnetic beads and vortex for 2 hours. Collect by ultrafiltration for later use.

[0069] 5. Incubate the magnetic bead nanoparticles (0.01 mg) prepared in step 3 with the antibody (0.045 mg) prepared in step 4 for 20–60 min to allow the antibody to couple with the magnetic nanoparticles. Collect the antibody-labeled nanoparticles by high-speed centrifugation and redisperse the antibody-nanoparticle conjugate in physiological buffer solution. A schematic diagram of the magnetic bead preparation process is shown below. Figure 1 As shown in the figure. The particle size distribution graph and potential comparison diagram before and after PEG grafting of magnetic beads are shown in the figure. Figure 2 , Figure 3 As shown in the electron microscope images, the ungrafted antibody nanoparticles are spherical with an average particle size of 42.5 nm and a relative standard deviation of 10%, exhibiting good monodispersity. The antibody-grafted magnetic beads have an average particle size of 50.4 nm and a relative standard deviation of 10%, also showing good monodispersity.

[0070] Meanwhile, CD8 antibody magnetic beads were prepared according to the methods and conditions in steps 1-5, except that CD4 antibody was replaced with CD8 antibody.

[0071] Example 2: Preparation of magnetic beads

[0072] Similar to Example 1, the difference is that in step 1, 20 ml (0.24 mmol) of 12 mM FeCl3·6H2O solution will be measured with a pipette, 2.8 g of FeCl2·4H2O (14 mmol) will be weighed with an analytical balance, and 10 g (0.25 mmol) of dextran will be weighed.

[0073] TEM images of magnetic beads, particle size distribution before and after PEG grafting, and potential comparison diagrams are shown below. Figure 4 , Figure 5 As shown in the electron microscope images, the ungrafted antibody nanoparticles are spherical with an average particle size of 173.4 nm and a relative standard deviation of 10%, exhibiting good monodispersity. The antibody-grafted magnetic beads have an average particle size of 195.2 nm and a relative standard deviation of 10%, also exhibiting good monodispersity.

[0074] Example 3: Preparation of magnetic beads

[0075] Similar to Example 1, the difference is that the proportions of each component are different; in step 1, 2.5 ml (0.03 mmol) of 12 mM FeCl3·6H2O solution will be measured with a pipette, 0.35 g of FeCl2·4H2O (1.75 mmol) and 2.5 g (0.0625 mmol) of dextran will be weighed with an analytical balance.

[0076] TEM images of magnetic beads, particle size distribution before and after PEG grafting, and potential comparison diagrams are shown below. Figure 6 , Figure 7 As shown in the electron microscope images, the ungrafted antibody nanoparticles are spherical with an average particle size of 24.3 nm and a relative standard deviation of 10%, exhibiting good monodispersity. The antibody-grafted magnetic beads have an average particle size of 46.3 nm and a relative standard deviation of 10%, also exhibiting good monodispersity.

[0077] The results above show that as the ratio of iron oxide to polysaccharide increases, the particle size of magnetic bead nanoparticles gradually increases.

[0078] Example 4: Preparation of magnetic beads

[0079] Similar to Example 1, except that the mass of CD4 antibody or CD8 antibody in step 5 is 0.03 mg.

[0080] Example 5: Preparation of magnetic beads

[0081] Similar to Example 1, except that the mass of CD4 antibody or CD8 antibody in step 5 is 0.07 mg.

[0082] Example 6: Toxicity Detection of Magnetic Beads

[0083] The magnetic beads prepared in Example 1 were subjected to cytotoxicity testing, and the results are as follows: Figure 8 As shown.

[0084] The results showed that the magnetic beads were essentially non-toxic to cells.

[0085] Example 7: Solubility of Magnetic Beads

[0086] The CD4 antibody magnetic bead nanoparticle dry powder sample prepared in Example 1 was dissolved in physiological buffer solution, and the solubility was detected to be 10 g / L to 60 g / L. The resulting solution did not precipitate after being left for half a year.

[0087] The physiological buffer is phosphate-buffered saline (PBS), sterile phosphate-buffered saline (D-PBS), Hank's balanced salt solution (HBSS), or Earle's balanced salt solution (EBSS).

[0088] Example 8: Detection of the trapping ability of magnetic beads

[0089] Lymphocytes were captured by magnetic bead sorting flow cytometry using magnetic beads prepared in Examples 1, 4, and 5.

[0090] Specific steps: First, perform T-cell lymphocyte counting, followed by centrifugation to collect cells. The centrifugation parameters for human cells were set to 300g / 10min, and for mouse cells to 400g / 10min. After centrifugation, the cells were resuspended. Calculations showed that 80μl corresponds to 10... 7 Cells were collected. Magnetic beads (prepared in Example 1, commercially available magnetic beads) were then added, following the instructions for commercially available magnetic beads. The cells were incubated at 4°C for 20 min. Cells were then washed with 1 ml of PBE solution and collected by centrifugation at 300 g / 10 min. After centrifugation, 500 μl of PBE solution was added to resuspend the cells. The column was then rinsed. After rinsing, the sample was added, and the column was washed three times. The separation tube was then separated from the magnetic poles, and PBE solution was added to wash off positive cells. Flow cytometry antibody was then added and incubated for 15 min. Finally, paraformaldehyde was added to fix the cells, and cell separation was performed using flow cytometry. The capture rate of the magnetic beads was determined by the proportion of cells in the Q2 region.

[0091] The results are as follows Figure 9-11 As shown in the results, the capture rate of CD4 antibody magnetic beads in Example 1 was 98.4%, and the capture rate of CD8 antibody magnetic beads was 80.4%. In Example 4, when the ratio of CD4 antibody or CD8 antibody to magnetic bead nanoparticles was 3:1, the capture rate was 33.0% or 11.3%. In Example 5, when the ratio of CD4 antibody or CD8 antibody to magnetic bead nanoparticles was 7:1, the capture rate was 39.4% or 19.4%. Therefore, it can be seen that when the ratio of antibody to magnetic bead nanoparticles is between 3:1 and 7:1, the magnetic beads have excellent capture ability; and when the ratio of antibody to magnetic bead nanoparticles is 4.5:1 (Example 1), the capture ability of the magnetic beads is the best.

[0092] Example 9: Magnetic beads for MRI imaging

[0093] The magnetic beads prepared in Example 1 were used to perform in vivo MRI imaging on 4T1 subcutaneous tumor-bearing female BALB / c mice.

[0094] Specifically, mice were injected via tail vein with Fe3O4@DEX particles (based on Fe3O4 concentration) at a dose of 15 mg Fe3O4 per kilogram of mouse body weight. During imaging, the mice were placed on a temperature-controlled animal bed and anesthetized by inhaling oxygen mixed with 2% isoflurane (600 mL / min). In vivo tracing results are as follows: Figure 12 As shown.

[0095] Test case

[0096] Procedure: First, T lymphocytes were counted, followed by centrifugation to collect the cells. The centrifugation parameters for human cells were set to 300g / 10min, and for mouse cells to 400g / 10min. After centrifugation, the cells were resuspended. 80μl corresponded to 10... 7 Cells were collected. Magnetic beads (Exp beads prepared in Example 1 and M commercial magnetic beads) were then added, following the instructions for the M commercial magnetic beads (Mitteni). The cells were incubated at 4°C for 20 min, followed by washing with 1 ml of PBE solution. Cells were then collected by centrifugation at 300 g / 10 min. After centrifugation, 500 μl of PBE solution was added to resuspend the cells. The column was then rinsed. After rinsing, the sample was added, and the column was washed three times. The separation tube was then separated from the magnetic poles, and PBE solution was added to wash off positive cells. Flow cytometry antibodies were then added and incubated for 15 min. Finally, paraformaldehyde was added to fix the cells, and cell separation was performed using a flow cytometer.

[0097] The results are as follows Figure 13 As shown in the results, the Fe3O4@DEX magnetic beads prepared in Example 1 have a similar capture ability to CD4 lymphocytes as the commercial magnetic beads group; however, for CD8 lymphocytes, the Fe3O4@DEX magnetic beads prepared in Example 1 have a much better capture ability than the commercial magnetic beads group.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A magnetic bead for T cell extraction, characterized in that: The magnetic beads comprise components of the following formula: A-L-B, wherein A is an epoxy magnetic bead nanoparticle, B is a thiolated T cell antibody, and L is a linking molecule, which is covalently linked to the epoxy group of A and the thiol group of B, respectively.

2. The magnetic beads for T cell extraction according to claim 1, wherein: The epoxy magnetic bead nanoparticle takes a magnetic metal oxide as a core, and the surface of the magnetic metal oxide is coated with polysaccharide, and an epoxy group is modified on the surface of the polysaccharide.

3. The magnetic beads for T cell extraction according to claim 1, wherein: The thiolated T cell antibody takes a T cell antibody as a core, and a thiol group is modified on the surface of the T cell antibody. Preferably, the T cell antibody comprises a CD4 antibody or a CD8 antibody.

4. The magnetic beads for T cell extraction of claim 1, wherein: The linking molecule comprises a biocompatible polymer with an active group and a complexing group. Preferably, the biocompatible polymer comprises one of linear or branched polyethylene glycol (PEG), and a block copolymer of linear or branched polyethylene glycol and polyacrylic acid (PAA), polymethacrylic acid (PMA), polyethylene amine (PEI), polyproline, polylysine, poly-leucine, polyglutamic acid, polyaspartic acid, or polylactic acid (PLA). Preferably, the active group is covalently linked to the thiol group of the T cell antibody, and the complexing group is covalently linked to the epoxy group of the magnetic bead nanoparticle. Preferably, the active group comprises a maleimide group, an alkyne group, an alkenyl group, an azido group, a cyano group, an enol group, or an enol ether group, and more preferably a maleimide group. Preferably, the complexing group comprises a carboxyl group, an amine group, a hydroxyl group, a thiol group, or a phosphoric acid group, and more preferably an amine group. Preferably, the biocompatible polymer comprises NHS-PEG-MAL (2k) or NHS-PEG (550).

5. The magnetic beads for T cell extraction of claim 1, wherein: The mass ratio of the linking molecule, the epoxy magnetic bead nanoparticle, and the thiolated T cell antibody is 25-4:1:10-2, and preferably 15-7:1:6-3.

6. A method for preparing magnetic beads for T cell extraction, characterized by, The method comprises covalently linking the epoxy magnetic bead nanoparticle and the thiolated T cell antibody by using the linking molecule.

7. The method for preparing magnetic beads for T cell extraction as described in claim 6, characterized in that, The preparation method of the epoxy bead nanoparticle comprises: 1) dissolving a magnetic metal compound and polysaccharide in water and reacting under an inert gas to obtain a polysaccharide-coated magnetic metal oxide solution; and 2) adding a basic solution and a molecule with an epoxy group in step 1) to react to obtain an epoxy magnetic bead nanoparticle. Preferably, the magnetic metal compound comprises a hydrated and anhydrous chloride salt, nitrate salt, and / or sulfate salt of a magnetic metal element, and the paramagnetic metal element comprises, but is not limited to, iron, gadolinium, neodymium, promethium, samarium, europium, terbium, dysprosium, holmium, erbium, and thulium, and more preferably iron. Preferably, the magnetic metal compound comprises ferric chloride and ferrous chloride. Preferably, the magnetic metal oxide comprises ferroferric oxide and ferrous oxide. Preferably, the polysaccharide comprises dextran. Preferably, the molar ratio of the magnetic metal compound to the polysaccharide is 10:1-100:1, and more preferably 20:1-75:

1. Preferably, the concentration of the magnetic metal compound is 0.001 mol / L-0.2 mol / L, and more preferably 0.01-0.1 mol / L. Preferably, in step 1), the reaction temperature is controlled at 25-40℃, preferably 25-35℃ under inert gas; the reaction time is 0.2-5 hours, preferably 0.5-2.5 hours; Preferably, the basic solution comprises sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia water; Preferably, the volume of the basic solution added is 0.01-0.2 times the volume of the polysaccharide-coated magnetic metal oxide solution; Preferably, the molecule with epoxy group comprises epoxy chloropropane; Preferably, the concentration of the molecule with epoxy group is 0.003 mol / L-0.8 mol / L, preferably the concentration is 0.03-0.6 mol / L.

8. The method for preparing magnetic beads for T cell extraction as described in claim 6, characterized in that, The preparation method of the thiolated T cell antibody comprises: reacting a thiolating agent and a T cell antibody to obtain a thiolated T cell antibody; Preferably, the thiolating agent comprises thioacetic acid, hydrogen sulfide, thiolate (such as sodium thiocyanate, potassium thiocyanate or TCEP), acetylthiol, benzyl mercaptoacetate; Preferably, the reaction time is 15-45 min.

9. The method for preparing magnetic beads for T cell extraction as described in claim 6, characterized in that, The method comprises the following steps: S1 covalently linking a linking molecule and an epoxy magnetic bead nanoparticle; S2 adding a thiolated T cell antibody in step S1 to react to obtain the magnetic bead; Preferably, in step S1, the covalent linking time is 1-100 hours, preferably 2-40 hours; Preferably, in step S2, the reaction time is 20-60 min; Preferably, the mass ratio of the thiolated T cell antibody to the epoxy magnetic bead nanoparticle is 3:1-7:

1.

10. Use of the magnetic bead of any one of claims 1-5 in the preparation of one or more application reagents: 1) T cell extraction, separation and screening; 2) in vivo tracing of lymph T cells; 3) evaluation and monitoring of CAR-T therapy effect; 4) early monitoring of tumor metastasis.

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