Antibody-grafted polyrotaxane hydrogel as well as preparation method and application thereof

Abstract

The invention discloses polyrotaxane hydrogel grafted with an antibody as well as a preparation method and application of the polyrotaxane hydrogel. The preparation method comprises the following steps: firstly synthesizing methacrylic acid modified polyrotaxane PR or Locked PR, and then modifying an upper antibody through a Michael addition reaction, so as to obtain ring-unlocked polyrotaxane hydrogel (PR hydr.) and ring-locked polyrotaxane hydrogel (Locked PR hydr.). The two hydrogels prepared in the invention drive the antibody to do free motion through the dynamic nature of the alpha-cyclodextrin on the rotaxane structure, so as to enhance the interaction between the antibody and a T cell antigen recognition receptor (TCR), thereby greatly improving the activation efficiency and amplification efficiency of T cells, and providing an effective way for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering materials, and particularly relates to a grafted antibody polyrotaxane hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, breakthroughs have been made in tumor immunotherapy, which has become the fourth major pillar of tumor treatment after surgery, radiotherapy, and chemotherapy (Int. Rev. Cell Mol. Biol., 2018, 341: 277-362). Generally speaking, tumor immunotherapy strategies mainly include four categories: adoptive cell immunotherapy, immune checkpoint inhibitor therapy, tumor vaccine therapy, and non-specific immune stimulation therapy. Among them, adoptive T cell therapy is a highly promising tumor immunotherapy strategy, which refers to a cell immunotherapy method in which tumor-specific T cells are activated and expanded in vitro and then transfused into patients to kill tumors. However, there are obstacles in the in vitro expansion link: the low in vitro expansion efficiency of T cells is one of the key problems hindering the development of T cell therapy. The number of T cells required for treatment is very large (10 9 ~10 11 cells), and the currently reported amplification methods take 6-8 weeks to amplify the therapeutic dose of T cells. However, T cells will undergo phenotypic changes or loss during the long-term amplification cycle, affecting the immunotherapy function of T cells (J. Immunother. Cancer, 2014, 2: 1-10); in vivo, T cells are stimulated by antigen-presenting cells and activated and amplified. However, directly using the antigen-presenting cells of patients to activate and amplify T cells faces two difficulties: First, the number of antigen-presenting cells in tumor patients is very small, making it difficult to amplify T cells to the therapeutic dose; second, directly using the antigen specificity presented by antigen-presenting cells to stimulate T cells often leads to immunosuppression, and then makes it difficult for T cells to exert their tumor-killing effect (Cell, 2014, 157: 357-368). Currently, researchers mainly focus on biomaterials, and through ingenious structural design, biomaterials are made to have immune activation functions to improve the in vitro expansion efficiency of T cells. Clinically, magnetic microspheres modified with aCD3 and aCD28 on the surface are mainly used to amplify CD8 +T cells are then reinfused into the patient's body to achieve the goal of killing tumors. However, the amplification efficiency of such magnetic microspheres is low, and it is difficult to amplify a therapeutic dose of T cells in a short period. In addition, it is difficult to completely separate the magnetic microspheres after amplification, and the residual magnetic microspheres will cause severe inflammatory reactions in the human body. To develop biomaterials that take into account both biosafety and amplification efficiency, researchers usually adopt a bionic strategy, designing various biomaterials by simulating the antigen presentation signals and cell membrane shapes of antigen-presenting cells, such as magnetic nanoparticles, silica microspheres, silica microrods, hydrogels and other materials. Although the efficiency of these biomaterials for in vitro amplification of T cells has been improved to some extent, it still takes about 14 days of amplification cycle to obtain a therapeutic dose of T cells. Therefore, the amplification efficiency needs to be further improved.

[0003] In terms of bionics, although they provide the three key signals required to activate T cells, none of them can simulate the dynamic presentation method of antigen-presenting antigen to T cells. We believe that this is the key reason why their amplification efficiency cannot be improved significantly. The dynamics during antigen presentation mainly refers to the fluidity of the cell membrane of antigen-presenting cells. This dynamics can provide biomechanical forces for the TCR (T cell antigen recognition receptor) / pMHC-I (major histocompatibility complex class I) complex, closely affecting the interaction strength of the TCR / pMHC-I complex, and further affecting the recognition efficiency of TCR for tumor antigens. This is also one of the core issues in tumor immunology (Nature, 2003, 423: 190-193). In recent years, biologists have discovered and confirmed that the TCR receptor is not only responsible for antigen recognition, but also an important mechanical sensor that can regulate the biological functions of T cells by sensing external forces (Progress in Biochemistry and Biophysics, 2021, 48: 637-645). Therefore, it is necessary to develop a biomaterial that can improve the amplification efficiency of T cells. Summary of the Invention

[0004] The primary object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a grafted antibody polyrotaxane hydrogel.

[0005] Another object of the present invention is to provide the application of the grafted antibody polyrotaxane hydrogel.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A grafted antibody polyrotaxane hydrogel is at least one of an unclosed polyrotaxane hydrogel PR hydr. (high mobility) and a closed polyrotaxane hydrogel Locked PR hydr. (low mobility); it is prepared by the following method:

[0008] S1. Synthesize the closed polyrotaxane Locked PR-ad

[0009] Dissolve adamantane-capped polyrotaxane (PR-ad) in an alkaline solution, heat it to 40 - 70 °C, add epichlorohydrin dropwise, stir and react. After the reaction is completed, dialyze and freeze-dry to obtain Locked PR-ad;

[0010] S2. Synthesize hydroxypropylated polyrotaxane PPG-PR-ad or Locked PPG-PR-ad

[0011] Dissolve adamantane-capped polyrotaxane (PR-ad) or Locked PR-ad obtained in step S1 in an alkaline solution, add propylene oxide dropwise under an ice bath, stir and react. After the reaction is completed, dialyze and freeze-dry to obtain PPG-PR-ad or Locked PPG-PR-ad;

[0012] S3. Synthesize methacrylic acid-modified polyrotaxane PR or Locked PR

[0013] Dissolve PPG-PR-ad or Locked PPG-PR-ad obtained in step S2, dibutyltin dilaurate (DBTDL), and 2,6-di-tert-butyl-p-cresol (BHT, inhibitor) in dimethyl sulfoxide (DMSO) to obtain a polyrotaxane mixed solution; dissolve isocyanatoethyl methacrylate in dimethyl sulfoxide (DMSO), and then drop it into the polyrotaxane mixed solution, and react at 37 °C - 60 °C. After the reaction is completed, purify and dry the product to obtain PR or Locked PR;

[0014] S4. Prepare antibody-polyrotaxane Ab-PR or Locked Ab-PR

[0015] Dissolve tris(2-hydroxyethyl)phosphine (TCEP) in water to obtain an aqueous TCEP solution; then add an antibody solution, mix well and carry out a reduction reaction under sealed conditions at 4 - 37 °C (preferably 4 °C). After the reaction is completed, ultrafilter and wash by centrifugation to obtain a reduced thiol-antibody solution; dissolve PR or Locked PR obtained in step S3 in a buffer solution to obtain a polyrotaxane solution; then mix the reduced thiol-antibody solution and the polyrotaxane solution evenly and react at 4 - 25 °C (preferably 4 °C) to obtain an antibody-polyrotaxane Ab-PR or Locked Ab-PR solution;

[0016] S5. Prepare antibody-polyrotaxane hydrogel PR hydr. or Locked PR hydr.

[0017] Dissolve the disulfhydryl-terminated polyethylene glycol (SH-PEG-SH) in a buffer solution with a pH of 7.4 to 9.0, and then add it to the antibody-polyrotaxane Ab-PR or Locked Ab-PR solution obtained in step S4. React at 4 to 25 °C (preferably 4 °C). After the reaction is completed, immerse the obtained gel in water or a buffer solution to remove unreacted antibodies, polyrotaxanes, and mercapto polyethylene glycol, obtaining an uncyclized polyrotaxane hydrogel PR hydr. or a cyclized polyrotaxane hydrogel Locked PR hydr., that is, the grafted antibody polyrotaxane hydrogel.

[0018] The threading rate of the adamantane-terminated polyrotaxane (PR-ad) described in steps S1 and S2 is approximately 22% (each α-CD completely coats two polyethylene glycol repeating units), that is, there are approximately 88 α-CDs on each polyrotaxane molecular chain; preferably, it is prepared by the following method:

[0019] (1) Synthesize carboxyl-terminated polyethylene glycol (PEG(COOH) 2 )

[0020] Dissolve polyethylene glycol in water, add 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and sodium bromide and stir to dissolve. Then add sodium hypochlorite solution to adjust the pH of the reaction solution to 10 to 11, stir and react. After the reaction is completed, add ethanol to quench the reaction, and then add hydrochloric acid solution to adjust the pH of the reaction solution < 2. Extract the reaction solution with dichloromethane, concentrate by rotary evaporation, and then drop it into ether. Collect the white precipitate and dry it to obtain PEG(COOH). 2 ;

[0021] (2) Synthesize pseudo-polyrotaxane pseudo-PR

[0022] Dissolve the PEG(COOH) obtained in step (1) 2 and α-cyclodextrin (α-CD) in hot water at 50 to 70 °C respectively to obtain an aqueous solution of PEG(COOH) 2 and an aqueous solution of α-CD; then drop the aqueous solution of PEG(COOH) 2 into the aqueous solution of α-CD heated to 60 ± 5 °C, stir vigorously and react. After the reaction is completed, freeze-dry to obtain pseudo-PR;

[0023] (3) Synthesize adamantane-terminated polyrotaxane PR-ad

[0024] Mix the pseudo-PR, 1-adamantylamine, Carter's condensing agent (benzotriazole-1-tri(trimetylamino)-hexafluorophosphate, BOP), and N,N-diisopropylethylamine (DIPEA) obtained in step (2) uniformly, then add N,N-dimethylformamide (DMF) dropwise until a paste is formed, stir and react at 4 °C, and after the reaction is completed, wash and freeze-dry to obtain PR-ad.

[0025] The weight-average molecular weight of the polyethylene glycol described in step (1) is 1 k to 50 k; preferably 35 K.

[0026] The amount of water used in step (1) is calculated as 20 - 50 mL of water per gram of polyethylene glycol; preferably calculated as 30 mL of water per gram of polyethylene glycol.

[0027] The water described in step (1) is preferably pure water.

[0028] The molar ratio of the polyethylene glycol, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and sodium bromide described in step (1) is 0.3:1.00 - 3.00:30.0 - 50.0; preferably 0.3:2.30:36.7.

[0029] The available chlorine content of the sodium hypochlorite solution described in step (1) > 5.0% (volume percentage).

[0030] The conditions for the stirring reaction in step (1) are: stir and react at room temperature for 10 - 20 min; preferably: stir and react at room temperature for 15 min.

[0031] The number of times of dichloromethane extraction in step (1) is preferably more than 3 times.

[0032] The conditions for drying in step (1) are: vacuum dry at 25 - 80 °C for 5 - 24 h; preferably: vacuum dry at 60 °C for 12 h.

[0033] The mass ratio of α-cyclodextrin (α-CD) to PEG(COOH) described in step (2) 2 is 4 - 8:1; preferably 4:1.

[0034] The concentration of the α-cyclodextrin (α-CD) aqueous solution described in step (2) is 2% - 10% by mass; preferably 2.4% by mass.

[0035] The PEG(COOH) 2 aqueous solution concentration described in step (2) is 1% - 10% by mass; preferably 1.2% by mass.

[0036] The stirring reaction time described in step (2) is 2 to 24 h; preferably 12 h.

[0037] In step (3), the molar ratio of the pseudo - rotaxane pseudo - PR, 1 - adamantylamine, the coupling agent (benzotriazole - 1 - tris (trimethylammonium) - trifluorophosphate, BOP), and N,N - diisopropylethylamine (DIPEA) is 1:50 - 150:5 - 20:5 - 20; preferably 1:100:10:10.

[0038] The stirring reaction time described in step (3) is 10 to 24 h; preferably 12 h.

[0039] The washing described in step (3) is carried out by washing successively with a mixed solution of N,N - dimethylformamide (DMF) and methanol, methanol, and hot water; preferably by washing successively with a mixed solution of N,N - dimethylformamide (DMF) and methanol, methanol, and hot water more than 2 times.

[0040] In the mixed solution of N,N - dimethylformamide (DMF) and methanol, the volume ratio of N,N - dimethylformamide (DMF) to methanol is 1:1.

[0041] In step S1, the mass ratio of the adamantane - capped polyrotaxane (PR - ad) to epichlorohydrin is 1:1 - 1.1.

[0042] In step S1, the concentration of the adamantane - capped polyrotaxane (PR - ad) in the reaction system is 1% - 8% by mass; preferably 6% by mass.

[0043] The basic solution described in steps S1 and S2 is an inorganic base solution or an organic base solution; preferably at least one of sodium hydroxide solution, potassium hydroxide solution, and triethylamine solution; more preferably sodium hydroxide solution.

[0044] The concentration of the basic solution described in steps S1 and S2 is 0.5 - 1.5 mol / L; preferably 1 mol / L.

[0045] The amount of the basic solution described in step S1 is calculated based on 14 - 16 mL of the basic solution per gram of PR - ad.

[0046] In step S1, the molar ratio of the base in the basic solution to the α - cyclodextrin (α - CD) is 20 - 40:1 (i.e., the molar amount of the base fed in the basic solution is 20 - 40 times the molar amount of α - CD); preferably 30:1.

[0047] The molar ratio of epichlorohydrin to the α-cyclodextrin (α-CD) described in step S1 is 8 to 35:1 (i.e., the molar amount of epichlorohydrin fed is 8 to 35 times the molar amount of α-CD); preferably 16:1. If the molar ratio of propylene oxide to α-cyclodextrin is too low, incomplete cross-linking of adjacent cyclodextrins will occur, and the movement restriction of cyclodextrin will be insufficient. If this molar ratio is too high, excessive cross-linking between the molecular chains of the polyrotaxane will occur, resulting in a significant reduction in the yield.

[0048] The temperature of the stirring reaction described in step S1 is preferably 50 to 60 °C; more preferably 50 °C.

[0049] The time of the stirring reaction described in step S1 is 6 to 48 h; preferably 24 h.

[0050] The dialysis described in step S1 is carried out using a dialysis bag with a molecular weight cut-off of 1000 to 2000 Da; preferably, dialysis is carried out using a dialysis bag with a molecular weight cut-off of 2000 Da for 48 to 72 h, and the water is changed every 3 to 6 h.

[0051] The dialysis liquid used for dialysis in steps S1 and S2 is deionized water.

[0052] The mass ratio of the adamantane-capped polyrotaxane (PR-ad) to propylene oxide described in step S2 is 1 to 1.4:4 to 5; preferably 1:5.

[0053] The mass ratio of the Locked PR-ad to propylene oxide described in step S2 is 1 to 4:4 to 5; preferably 4:5.

[0054] The molar ratio of propylene oxide to the α-cyclodextrin (α-CD) described in step S2 is 20 to 150:1 (i.e., the molar amount of propylene oxide fed is 20 to 150 times the molar amount of α-CD); preferably 38 to 114:1.

[0055] The dissolution described in step S2 is stirring dissolution; preferably, it is stirred for 10 min in an ice bath to dissolve it, and then propylene oxide is added dropwise.

[0056] The time of the stirring reaction described in step S2 is 6 to 24 h; preferably 12 h (the stirring reaction is overnight).

[0057] The dialysis described in step S2 is carried out using a dialysis bag with a molecular weight cut-off of 1000 to 2000 Da; preferably, dialysis is carried out using a dialysis bag with a molecular weight cut-off of 2000 Da for 12 to 28 h.

[0058] The concentration of the polyrotaxane mixed solution described in step S3 is 1% to 4% by mass; preferably 3% to 4% by mass.

[0059] In the PPG-PR-ad or Locked PPG-PR-ad structure described in step S3, the molar ratio of α-cyclodextrin, dibutyltin dilaurate (DBTDL), 2,6-di-tert-butyl-p-cresol (BHT, polymerization inhibitor), and isocyanatoethyl methacrylate is 1: 0.01-0.05: 0.003-0.02: 0.1-2; preferably 1: 0.034-0.043: 0.011-0.02: 0.23-0.45.

[0060] The purification described in step S3 is carried out by methods such as organic solvent precipitation, dialysis, or extraction; preferably by organic solvent precipitation; wherein, the organic solvent is at least one of acetone, ether, and n-hexane; more preferably by purification with ice acetone (washing with acetone more than 2 times).

[0061] The reaction temperature described in step S3 is preferably 40°C to 50°C; more preferably 45°C to 50°C.

[0062] In step S4, the operation of preparing the reduced thiol-antibody solution is carried out in a sterile environment.

[0063] The water described in step S4 is preferably sterile water.

[0064] The concentration of the TCEP aqueous solution described in step S4 is 10-50 mmol / L; preferably 10-30 mmol / L; more preferably 12-20 mmol / L.

[0065] The concentration of the antibody solution described in step S4 is 0.08-10 μg / μL; preferably 0.08 μg / μL.

[0066] The antibody described in step S4 is an antibody or polypeptide that can activate T cells; preferably aCD3 and aCD28; more preferably an antibody obtained by mixing aCD3 and aCD28 in a mass ratio of 1-2: 1-2; even more preferably an antibody obtained by mixing aCD3 and aCD28 in a mass ratio of 1: 1.

[0067] In step S4, the antibody concentration in the reduction reaction system is 0-1000 μg / mL (excluding 0).

[0068] The volume ratio of the TCEP aqueous solution and the antibody solution described in step S4 is 1: 1.

[0069] The time of the reduction reaction described in step S4 is 0.5-2 h; more preferably 1-2 h; even more preferably 1 h.

[0070] The ultrafiltration described in step S4 is carried out using a 50 kDa ultrafiltration tube.

[0071] The centrifugation conditions described in step S4 are: centrifugation at 800 g for 10 min.

[0072] The washing described in step S4 is performed using a PBS (pH 7.4) buffer solution; preferably, it is washed with a PBS (pH 7.4) buffer solution more than 2 times.

[0073] The concentration of the thiol-antibody solution described in step S4 is 0.01 - 10 μg / μL; preferably, it is 0.096 μg / μL.

[0074] The buffer solution described in step S4 is a Tris-HCl buffer solution with a pH of 8.0 - 9.0; preferably, it is a Tris-HCl buffer solution with a pH of 8.8.

[0075] The mass concentration of the polyrotaxane solution (PR solution or Locked PR solution) described in step S4 is 10 - 30% (preferably 28 - 30%), and the specific situation depends on the modification rate of the methacrylic acid double bond on the polyrotaxane structure. The double bond concentration in the solution needs to satisfy ≥0.5 mol / L and ≤2 mol / L; preferably, it is 1.0 mol / L.

[0076] The mass ratio of the antibody in the thiol-antibody solution to the polyrotaxane (PR or Locked PR) in the polyrotaxane solution described in step S4 is 1 - 4:216667.

[0077] The mixing evenly described in step S4 is preferably achieved by ultrasonic mixing. The ultrasonic time should not be too long. After stirring, it can be ultrasonicated for 5 - 10 min. An ice bag can be added to the ultrasonic water to maintain a low-temperature environment and avoid affecting the antibody activity.

[0078] In step S4, the reaction time (grafting antibody) of the thiol-antibody solution and the polyrotaxane solution is 2 - 24 h; preferably, it is 5 h.

[0079] The buffer solution described in step S5 is preferably a Tris-HCl buffer solution with a pH of 8.0 - 9.0 or a PBS buffer solution with a pH of 7.4.

[0080] The reaction time described in step S5 is 1 - 24 h; preferably, it is 12 h.

[0081] In step S5, the container for gel formation can be a 12-well plate, 24-well plate, 48-well plate, or 96-well plate.

[0082] The molecular weight of the bis-thiol-terminated polyethylene glycol (SH-PEG-SH) described in step S5 is 1 - 50 K; preferably, it is 5 k.

[0083] In step S5, the solution formed by dissolving the dithiol-terminated polyethylene glycol (SH-PEG-SH) in a buffer solution with a pH of 7.4 to 9.0 has a concentration of 97 to 99% by mass.

[0084] In step S5, the volume ratio of the dithiol-terminated polyethylene glycol (SH-PEG-SH) to the antibody-polyrotaxane Ab-PR (or Locked Ab-PR) is 1.5 to 2.0:1; preferably 1.7:1.

[0085] In step S5, soaking the obtained gel in a buffer solution means soaking the obtained gel in ice-cold PBS buffer solution for 1 hour, discarding the PBS buffer solution, and repeating the operation 3 to 5 times (preferably 4 times) to remove unreacted antibodies, polyrotaxanes, and mercapto-polyethylene glycol.

[0086] The polyrotaxane hydrogel grafted with antibodies described in step S5 can be stored short-term at 4°C and long-term at -20°C without repeated freezing and thawing.

[0087] The polyrotaxane hydrogel grafted with antibodies is used for in vitro expansion of CD8 + T cells (for non-disease diagnosis and treatment purposes).

[0088] The polyrotaxane hydrogel grafted with antibodies can stimulate CD8 + T cells to produce effector T cell subsets and can produce memory T cells; among them, the uncyclized polyrotaxane hydrogel PR hydr. can stimulate CD8 + T cells to produce effector T cell subsets, and the cyclized polyrotaxane hydrogel Locked PR hydr. can stimulate CD8 + T cells to produce relatively more memory T cells.

[0089] A method for in vitro expansion of CD8 + T cells, comprising the following steps:

[0090] I. Pretreatment of the polyrotaxane hydrogel

[0091] Soak the above polyrotaxane hydrogel grafted with antibodies in a PBS buffer solution containing 1% to 5% (v / v) penicillin-streptomycin (double antibody), soak (sterilize) for 1 to 5 h, then discard the PBS buffer solution, add a culture medium, soak at 4°C for 30 min to 2 h (preferably 2 h), and then discard the culture medium to obtain the pretreated polyrotaxane hydrogel (PR hydr. and / or Locked PR hydr.);

[0092] II. Expansion of CD8 + T cells

[0093] Add CD8+ The T cell suspension was added to the pretreated polyrotaxane hydrogel obtained in Step I, and the mixture was evenly mixed. The mixture was cultured at 37 °C and 5% CO 2 conditions. On the 3rd day of culture, a culture medium containing 1 - 1000 ng / mL of IL-2 with a volume 3 - 5 times (preferably 3 times) that of the original was added for continuous culture. On the 4th or 5th day of culture, a culture medium containing 1 - 1000 ng / mL of IL-2 with a volume 3 - 10 times (preferably 3 times) that of the original was added for continuous culture. The culture medium was changed every 2 - 3 days, and the cells were collected on the 7th - 14th day.

[0094] The dosage of penicillin - streptomycin (double antibody) described in Step I is preferably calculated according to its final concentration in the system being 2% by volume percentage. The higher the concentration of the double antibody, the shorter the required disinfection time, but too high a concentration is likely to cause the antibody to denature and inactivate.

[0095] The soaking (disinfection) time described in Step I is preferably 3 h.

[0096] The culture medium described in Steps I and II is preferably RPMI 1640 complete medium.

[0097] The CD8 + T cells described in Step II are preferably CD8 + T cells obtained from humans or animals; preferably CD8 + T cells obtained from mice, where the mice are preferably C57 mice at 8 - 11 weeks of age, and the spleen cells of mice at this age have better viability.

[0098] The CD8 + T cell suspension has a density of 0.5×10 6 ~5×10 6 cells / mL; preferably 3×10 6 cells / mL.

[0099] In Step II, the culture medium added in the first three days does not contain IL-2, so as to fully activate the CD8 + T cells on the gel. On the third day of culture, a culture medium containing IL-2 was added to assist in the expansion of T cells.

[0100] The concentration of IL-2 in the culture medium described in Step II is preferably 1000 ng / mL.

[0101] In Step II, the medium replacement method is to directly add multiple volumes of fresh complete medium. The multiple depends on the actual amplification situation and can be 3 to 5 times; on the 5th day, the medium replacement method is also to directly add multiple volumes of fresh complete medium, and the multiple can be 3 to 10 times; the amplification culture time can be extended to 10 to 14 days according to the actual growth status of the cells. However, when the in vitro culture of T cells exceeds 14 days, the activity significantly decreases; generally speaking, the medium is replaced every 2 to 3 days.

[0102] In Step II, CD8 is amplified + The culture containers for T cells can be 6-well plates, 12-well plates, 24-well plates, and 48-well plates.

[0103] The method for in vitro amplification of CD8 + T cells further includes the steps of further counting and / or identifying the cells after Step II.

[0104] The counting can be performed using a hemocytometer or a flow cytometer, and the cell amplification multiple can be further calculated therefrom.

[0105] The identification of the cells can be performed by staining and then testing and identifying with a flow cytometer.

[0106] In the present invention, the free movement of α-cyclodextrin driving the antibody within a certain range can increase the probability of contacting TCR (T cell antigen recognition receptor), thereby improving the activation efficiency of T cells; furthermore, when the antibody (antigen peptide) on the polyrotaxane forms an antigen-antibody complex with TCR recognition, the rotation and sliding of cyclodextrin on the polyrotaxane provide an appropriate shear force for the complex, and this shear force generates an inverse lock-key effect, enhancing the interaction of the antigen peptide / TCR complex. This polyrotaxane hydrogel (PR hydr.) greatly improves the activation efficiency of T cells. At the same time, in order to regulate the activation of T cells, we also crosslink adjacent α-cyclodextrins to weaken the free movement of α-cyclodextrin. This hydrogel with weakened dynamics is called locked ring or motion-restricted polyrotaxane hydrogel (Locked PR hydr.). Through experimental research, it is found that whether it is PR hydr. or LockedPR hydr., their amplification efficiencies are significantly better than the commercial amplification reagent Dynabeads, and the amplification efficiency of PR hydr. is better than that of Locked PR hydr. In addition, the CD8 + T cells activated by PR hydr. can produce more effector subsets, while the CD8 + T cells activated by LockedPR hydr. can produce more memory subsets. Therefore, in practical applications, different functional CD8s can be transfused according to the disease course of tumor patients +T cells to improve the efficiency of T cell immunotherapy, enabling timely inhibition of tumors and suppression of their recurrence.

[0107] The present invention has the following advantages and effects compared with the prior art:

[0108] (1) The present invention provides a grafted antibody unlocked polyrotaxane hydrogel (PR hydr., high mobility) and a locked polyrotaxane hydrogel (Locked PR hydr., low mobility). The grafted antibody polyrotaxane hydrogel includes an unlocked polyrotaxane hydrogel and a locked polyrotaxane hydrogel material. The two hydrogels of the present invention drive the antibody to move freely through the dynamics of α-cyclodextrin on their rotaxane structure, enhancing the interaction between the antibody and TCR, thereby greatly improving the activation efficiency and amplification efficiency of T cells, which is significantly superior to commercial amplification reagents.

[0109] (2) The grafted antibody polyrotaxane hydrogel in the present invention is composed of an α-CD / PEG type polyrotaxane derivative, thiol-polyethylene glycol, and an antibody. The antibody is modified in a relatively mild buffer solution through a Michael addition reaction. Then, the polyrotaxane modified with the antibody is crosslinked with thiolated polyethylene glycol through a Michael addition reaction to prepare the hydrogel. The synthesis conditions of the antibody-polyrotaxane and the hydrogel preparation conditions are green, mild, and simple. The prepared polyrotaxane has a regular structure and a low molecular dispersion coefficient, and is easy to regulate.

[0110] (3) The present invention utilizes the nucleophilic substitution reaction between epichlorohydrin and the hydroxyl groups on cyclodextrin to crosslink adjacent α-CDs on the polyethylene glycol axis to achieve the purpose of restricting its mobility. The degree of crosslinking of adjacent cyclodextrins can be controlled by adjusting the ratio. This method is efficient and controllable and can be used to regulate the dynamics of polyrotaxane.

[0111] (4) The present invention modifies the antibody on the cyclodextrin of polyrotaxane through click chemistry. The reaction conditions are mild, green, and efficient, and can well maintain the affinity of the antibody.

[0112] (5) The present invention prepares a polyrotaxane hydrogel by crosslinking the antibody-polyrotaxane and terminal thiol polyethylene glycol through a click reaction. The gelation conditions are mild and efficient.

[0113] (6) In terms of safety, compared with the traditional magnetic bead amplification method, the polyrotaxane hydrogel provided by the present invention is more non-toxic and safe. After magnetic bead amplification of T cells, a magnetic rack is needed for separation, and it is inevitable to have residual magnetic beads in the cells. The residual magnetic beads enter the human body through blood circulation and are likely to cause side reactions such as local embolism, inflammation, and allergy.

[0114] (7) In terms of the amplification effect, compared with traditional amplification methods, the two hydrogels provided by the present invention have a better amplification effect. The special dynamics provided by α-CD on the polyrotaxane for antibodies can greatly improve the activation efficiency of T cells. Moreover, polyrotaxanes with different mobilities can provide space for the free rotation and sliding of antibodies, increasing the recognition efficiency between antibodies and TCR receptors on the T cell membrane.

[0115] (8) In terms of the activated subsets, the CD8 + T cells activated by PR hydr. provided by the present invention can produce more effector memory subsets, which can be used for the rapid killing of tumor cells and effectively inhibit tumor growth; while the Locked PRhydr. activates CD8 + T cells can produce more central memory T cell subsets, which can achieve long-term surveillance of tumor cells to inhibit tumor recurrence.

[0116] (9) In terms of the activity after amplification, compared with traditional amplification methods, the T cells amplified by the two polyrotaxane hydrogels provided by the present invention have very good viability and do not contain any mycoplasma, endotoxin and other components. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 is a schematic diagram of the network structure of PR hydr. (in the figure, a, b, d, and e are the numbers of α-cyclodextrins participating in the cross-linking reaction on each polyrotaxane molecular axis, and the estimated values are all 4; c is the number of α-cyclodextrins participating in connecting antibodies on each polyrotaxane molecular axis, and the estimated value is 1).

[0118] Figure 2 is a schematic diagram of the network structure of Locked PR hydr. (in the figure, a, b, d, and e are the numbers of α-cyclodextrins participating in the cross-linking reaction on each polyrotaxane molecular axis, and the estimated values are all 4; c is the number of α-cyclodextrins participating in connecting antibodies on each polyrotaxane molecular axis, and the estimated value is 1).

[0119] Figure 3 is a schematic diagram of the synthesis process of PR and Locked PR.

[0120] Figure 4 is the nuclear magnetic resonance hydrogen spectrum of PR prepared in Example 1.

[0121] Figure 5 is the nuclear magnetic resonance hydrogen spectrum of Locked PR prepared in Example 2.

[0122] Figure 6 is a physical picture of PR hydr. prepared in Example 4 and Locked PR hydr. prepared in Example 5.

[0123] Figure 7 It is a graph showing the antibody grafting efficiency results of PR hydr. prepared in Example 4.

[0124] Figure 8 It is a flowchart of the operation of amplifying CD8 + T cells using a polyrotaxane hydrogel (the temperature and time on the horizontal line in Step ⑦ refer to the temperature and total duration of disinfecting the gel and washing the gel).

[0125] Figure 9 It is a statistical chart of the cytotoxicity of the PR hydr. group prepared in Example 4, the Locked PR hydr. group prepared in Example 5, the TCP group and the Dynabeads group in Step d of Example 7.

[0126] Figure 10 It is the graph of the amplification multiples of CD8 + T cells activated by PR dydr. prepared in Example 4 and Locekd PR hydr. prepared in Example 5 on different days.

[0127] Figure 11 It is the graph of the cell subsets produced by CD8 + T cells activated by PR dydr. prepared in Example 4 and Locekd PR hydr. prepared in Example 5. Detailed implementation manners

[0128] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test methods without specific experimental conditions in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0129] Example 1 Synthesis of PR

[0130] a. Synthesis of carboxyl-terminated polyethylene glycol (PEG(COOH) 2 )

[0131] Take 10.0 g of polyethylene glycol (weight-average molecular weight Mw = 35K) in a 500 mL conical flask, add 300 mL of pure water, and stir to dissolve it; successively add 344 mg of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) (M = 156 g / mol, 2.20 mmol) and 3.60 g of sodium bromide (M = 103 g / mol, 35.0 mmol), and stir to completely dissolve it; add 17.5 mL of sodium hypochlorite solution (available chlorine > 5.0%) to the reaction solution to adjust the pH of the reaction solution to 10 - 11, and stir at room temperature for 15 min; at the end of the reaction, add 10 mL of pure ethanol to quench the reaction, and then drop in an appropriate amount of hydrochloric acid solution to adjust the pH of the reaction solution < 2; extract the reaction solution with dichloromethane 3 times, 100 mL each time. Concentrate the collected dichloromethane solution to about 10 mL by rotary evaporation, drop it into ether, and collect the white precipitate; collect the white precipitate in a glass, seal it with filter paper, place it in a vacuum drying oven, and dry it under vacuum at 60 °C for 12 h. The obtained white or nearly white precipitate is PEG(COOH). 2 , and the yield is 95%.

[0132] b. Preparation of α-CD / PEG(COOH) 2 Pseudo-rotaxane pseudo-PR

[0133] Take 4.80 g of α-CD (α-cyclodextrin) and dissolve it in 20 mL of hot water at 50 - 70 °C (placed in a 100 mL reaction flask) to obtain an α-CD aqueous solution; take 1.20 g of PEG(COOH) 2 and dissolve it in 10 mL of hot water at 50 - 70 °C to obtain an aqueous solution of PEG(COOH). 2 Then heat the α-CD aqueous solution to 60 °C, and drop the aqueous solution of PEG(COOH) 2 into the α-CD aqueous solution while stirring vigorously. After reacting for 12 h, it can be seen that the reaction solution becomes a white paste. After freeze-drying, the obtained white solid is α-CD / PEG(COOH). 2 Pseudo-rotaxane pseudo-PR, and the yield is 99%.

[0134] c. Synthesis of adamantane-capped polyrotaxane PR-ad

[0135] Take 1.50 g (0.0315 mmol) of pseudo - rotaxane pseudo - PR, 0.480 g of 1 - adamantylamine (CAS No.: 768 - 94 - 5; M = 151 g / mol, 3.15 mmol), 0.139 g of Carter's condensing agent (benzotriazole - 1 - tris (trimethylamino) - hexafluorophosphate, BOP) (M = 442 g / mol, 0.315 mmol), and 0.0550 mL of N,N - diisopropylethylamine (DIPEA) (M = 129 g / mol, ρ = 0.742 g / mL, 0.316 mmol). Mix them evenly in a reaction flask, and while stirring, add an appropriate amount of N,N - dimethylformamide (DMF) dropwise until a muddy state is formed. Place the reaction device in a chromatography cabinet, maintain at 4 °C, and stir the reaction for 12 hours. At the end of the reaction, wash the reaction solution twice with a DMF / methanol mixed solution (volume ratio 1:1), methanol, and hot water (50 - 70 °C), centrifuge, collect the product precipitate, and freeze - dry to obtain a white solid product PR - ad with a yield of 60%.

[0136] d. Synthesis of hydroxypropylated polyrotaxane PPG - PR - ad

[0137] Take 1 g of PR - ad (Mw = 100 k, containing 0.01 mmol of PR - ad, 0.88 mmol of α - CD) and dissolve it in 20 mL of 1 M sodium hydroxide solution. Stir for 10 min in an ice bath, and drop - add 6 mL of pure propylene oxide solution (M = 50.08 g / mol, ρ = 0.830 g / mL, 100 mmol). Stir the reaction overnight. Purify the product by dialysis (cut - off molecular weight MWCO 2000 Da, dialysis solution is deionized water, dialysis time 12 - 28 h). The white solid obtained after freeze - drying is PPG - PR - ad with a yield of 90%.

[0138] e. Synthesis of methacrylic acid - modified polyrotaxane MA - PPG - PR - ad (abbreviation: PR)

[0139] 1.3 g of PPG-PR-ad (Mw = 130 k, containing 0.01 mmol of PR-ad and 0.880 mmol of α-CD) was dissolved in 35 mL of ultradry dimethyl sulfoxide (DMSO) (the mass concentration of the polyrotaxane in the system was 3.3%). 0.02 mL of dibutyltin dilaurate (DBTDL) (M = 631.56 g / mol, ρ = 1.066 g / mL, 0.03 mmol) and 2.0 mg of 2,6-di-tert-butyl-p-cresol (BHT, inhibitor) (M = 220.35 g / mol, 0.01 mmol) were successively added and stirred until dissolved to obtain a polyrotaxane mixed solution. 60.0 mg of isocyanatoethyl methacrylate (M = 155.15 g / mol, 0.40 mmol) was dissolved in 1.0 mL of ultradry DMSO and added dropwise to the polyrotaxane mixed solution, and the reaction was carried out overnight at 50 °C. Among them, the molar ratio of α-cyclodextrin, DBTDL, BHT, and isocyanatoethyl methacrylate in the polyrotaxane structure was 1:0.034:0.011:0.45. At the end of the reaction, the reaction solution was dropped into ice acetone to obtain a sticky white solid precipitate, and the precipitate was washed twice with acetone. Vacuum drying at room temperature gave a nearly white viscous solid as PR with a yield of 80%. The synthesis reaction equation of the final product PR is as Figure 3 shown, and the 1H NMR characterization analysis of the product is as Figure 4 shown.

[0140] Example 2 Synthesis of Locked PR

[0141] a. Synthesis of carboxyl-terminated polyethylene glycol (PEG(COOH) 2 )

[0142] The synthesis method was the same as that in step a of Example 1.

[0143] b. Preparation of α-CD / PEG(COOH) 2 pseudo-rotaxane pseudo-PR

[0144] The synthesis method was the same as that in step b of Example 1.

[0145] c. Synthesis of adamantane-capped polyrotaxane PR-ad

[0146] The synthesis method was the same as that in step c of Example 1.

[0147] d. Synthesis of locked polyrotaxane Locked PR-ad

[0148] Take 2.0 g of PR-ad (Mw = 120 k, containing 0.02 mmol of PR-ad and 1.76 mmol of α-CD) and place it in a round-bottom flask. Add 34 mL of 20% (w / v) sodium hydroxide solution, raise the temperature to 50 °C, and stir to completely dissolve PR-ad. After the reactants are completely dissolved, slowly add 2.6 g of epichlorohydrin (M = 92.5 g / mol, 28.0 mmol) dropwise while stirring. The molar ratio of α-cyclodextrin to epichlorohydrin is approximately 1:16. After reacting for 24 hours, transfer the reaction solution to a dialysis bag (Mwco 2000 Da), use deionized water as the dialysis solution, and dialyze for 48 - 72 h, changing the water every 3 - 6 h. Freeze-dry to obtain the product Locked PR-ad with a yield of 70%.

[0149] e. Synthesis of hydroxypropylated polyrotaxane Locked PPG-PR-ad

[0150] Take 4.0 g of Locked PR-ad (Mw = 135 k, containing 0.04 mmol of PR-ad and 2.60 mmol of α-CD) and dissolve it in 24 mL of 1 M sodium hydroxide solution. Stir for 10 min in an ice bath, and slowly add 6.0 mL of pure propylene oxide solution (M = 50.08 g / mol, ρ = 0.830 g / mL, 100 mmol). Stir and react overnight. Purify the product by dialysis (MWCO 2000 Da, dialysis solution is deionized water, dialysis time 12 - 28 h). The white solid obtained after freeze-drying is Locked PPG-PR-ad with a yield of 90%.

[0151] f. Synthesis of methacrylic acid-modified polyrotaxane Locked MA-PPG-PR-ad (abbreviated as Locked PR)

[0152] Take 1.5 g of Locked PPG PR - ad (Mw = 150 k, containing 0.01 mmol of PR - ad and 0.880 mmol of α - CD) and dissolve it in 35 mL of ultra - dry DMSO (the mass concentration of the polyrotaxane is 3.8%). Sequentially add 0.02 mL of dibutyltin dilaurate (DBTDL) (M = 631.56 g / mol, ρ = 1.066 g / mL, 0.03 mmol) and 2.0 mg of 2,6 - di - tert - butyl - p - cresol (BHT, inhibitor) (M = 220.35 g / mol, 0.01 mmol), and stir to dissolve to obtain a polyrotaxane mixed solution. Take 30.0 mg of isocyanatoethyl methacrylate (M = 155.15 g / mol, 0.20 mmol) and dissolve it in 1.0 mL of ultra - dry DMSO, and drop it into the polyrotaxane mixed solution, and react at 50 °C overnight. Among them, the molar ratio of α - cyclodextrin, DBTDL, BHT, and isocyanatoethyl methacrylate in the polyrotaxane structure is 1:0.034:0.011:0.23. At the end of the reaction, drop the reaction solution into ice - acetone to obtain a sticky white solid precipitate, and wash the precipitate with acetone twice. Dry it under vacuum at room temperature to obtain a nearly white viscous solid, Locked PR, with a yield of 80%. The synthesis reaction equation of the final product Locked PR is as Figure 3 shown, and the 1H NMR characterization analysis of the product is as Figure 5 shown.

[0153] Example 3 Synthesis of PR

[0154] a. Synthesis of carboxyl - terminated polyethylene glycol (PEG(COOH) 2 )

[0155] The synthesis method is the same as step a of Example 1.

[0156] b. Preparation of α - CD / PEG(COOH) 2 pseudo - rotaxane pseudo - PR

[0157] The synthesis method is the same as step b of Example 1.

[0158] c. Synthesis of adamantane - capped polyrotaxane PR - ad

[0159] The synthesis method is the same as step c of Example 1.

[0160] d. Synthesis of hydroxypropylated polyrotaxane PPG - PR - ad

[0161] Dissolve 2.0 g of PR-ad (Mw = 100 k, containing 0.02 mmol of PR-ad and 1.60 mmol of α-CD) in 40 mL of 1 M sodium hydroxide solution, stir for 10 min in an ice bath, and dropwise add 10.0 mL of pure propylene oxide solution (M = 50.08 g / mol, ρ = 0.830 g / mL, 166 mmol), then stir the reaction overnight. Purify the product by dialysis (MWCO 2000 Da, dialysis solution is deionized water, dialysis time 12 - 48 h). The white solid obtained after freeze-drying is PPG-PR-ad, and the yield is 90%.

[0162] e. Synthesize methacrylic acid-modified polyrotaxane MA-PPG-PR-ad (abbreviation: PR)

[0163] Dissolve 1.5 g of PPG-PR-ad (Mw = 150 k, containing 0.01 mmol of PR-ad and 0.880 mmol of α-CD) in 35 mL of ultra-dry DMSO (mass concentration of polyrotaxane is 3.8%). Sequentially add 0.02 mL of dibutyltin dilaurate (DBTDL) (M = 631.56 g / mol, ρ = 1.066 g / mL, 0.038 mmol) and 4.0 mg of 2,6-di-tert-butyl-p-cresol (BHT, inhibitor) (M = 220.35 g / mol, 0.018 mmol), stir to dissolve to obtain a polyrotaxane mixed solution. Dissolve 60.0 mg of isocyanatoethyl methacrylate (M = 155.15 g / mol, 0.39 mmol) in 1.0 mL of ultra-dry DMSO, and drop it into the polyrotaxane mixed solution, then react at 50 °C overnight. Among them, the molar ratio of α-cyclodextrin, DBTDL, BHT and isocyanatoethyl methacrylate in the polyrotaxane structure is 1:0.043:0.02:0.44. At the end of the reaction, drop the reaction solution into ice acetone to obtain a sticky white solid precipitate, and wash the precipitate with acetone twice. Dry it under vacuum at room temperature to obtain a nearly white viscous solid as PR, with a yield of 75%. After characterization, its structural formula is the same as that in Example 1.

[0164] Example 4 Preparation of polyrotaxane hydrogel PR hydr.

[0165] a. Preparation of antibody-polyrotaxane Ab-PR

[0166] Reduced antibody: The following operations are carried out in a sterile environment. Take 5.0 mg of tris(2-hydroxyethyl)phosphine (TCEP) and dissolve it in 1.0 mL of sterile water to obtain an aqueous TCEP solution with a concentration of 20 mM. Take 120 μL each of aCD3 and aCD28 antibody solutions (Anti-mouse CD3 (abbreviated as aCD3), Clone: 145-2C11, Isotype: Armenian Hamster, BioXcell (USA); Anti-mouse CD28 (abbreviated as aCD28), Clone: 37.51, Isotype: Syrian, Biolegend (USA)) with a concentration of 0.08 μg / μL (the mass ratio of aCD3 and aCD28 is 1:1), mix them evenly with 240 μL of the aqueous TCEP solution, seal, place on a shaker, react at 4 °C for 1 hour, transfer the mixed solution to a 50 kDa ultrafiltration tube, centrifuge (800 g, 10 min), add sterile PBS (pH 7.4) solution to the inner tube of the ultrafiltration tube, mix evenly, centrifuge (800 g, 10 min), and repeat this washing operation twice. Finally, concentrate the antibody to a total volume of 100 μL with PBS (pH 7.4) to obtain a reduced antibody solution with a concentration of 0.096 μg / μL (i.e., a solution containing thiol-aCD3 and thiol-aCD28).

[0167] Grafted antibody: Take 17 μL of a 28% (mass concentration) polyrotaxane solution (PR synthesized in Example 1, dissolved in Tris-HCl with pH 8.8) in a 96-well polytetrafluoroethylene well plate, add 3 μL of the above-reduced antibody solution, mix evenly (mix evenly by ultrasonic method, the ultrasonic time should not be too long, it can be ultrasonicated for 5 - 10 min after stirring, an ice bag can be added to the ultrasonic water to keep the environment at a low temperature to avoid affecting the antibody activity; the same below), place at 4 °C and react for 5 hours to obtain an antibody-polyrotaxane solution.

[0168] b. Preparation of antibody-polyrotaxane hydrogel Ab-PR hydr. (abbreviated as PR hydr.)

[0169] Take 1.0 mg of bis-thiol-terminated polyethylene glycol (SH-PEG-SH) (Mw = 5k) and dissolve it in 10 μL of Tris-HCl (pH 8.8) solution, then add it to the above antibody-polyrotaxane solution, mix evenly by ultrasonic, place at 4 °C and react for 12 hours to obtain a polyrotaxane gel modified with an antibody. Immerse this gel in ice-cold PBS for 1 hour, discard the PBS, and repeat the operation 4 times to remove unreacted antibodies, polyrotaxanes, and thiol polyethylene glycol. The schematic diagram of the network structure of the obtained PR hydrogel (PR hydr.) is as shown in Figure 1 shown, and the physical object is as shown in Figure 6As shown. The antibody grafting efficiency of the hydrogel was measured by a conventional enzyme-linked immunosorbent assay (ELISA), as Figure 7 shown.

[0170] Example 5 Preparation of Locked Polyrotaxane Hydrogel Locked PR hydr.

[0171] a. Preparation of Locked Antibody-Polyrotaxane Locked Ab-PR

[0172] Reduction of antibody: The following operations were carried out in a sterile environment. 5.0 mg of tris(2-hydroxyethyl)phosphine (TCEP) was dissolved in 1.0 mL of sterile water at a concentration of 20 mM to obtain an aqueous TCEP solution. 120 μL each of aCD3 and aCD28 antibodies (the same as in Example 4) with a concentration of 0.08 μg / μL were taken and mixed evenly with 240 μL of the aqueous TCEP solution, sealed, placed on a shaker, and reacted at 4 °C for 1 hour. The mixed solution was transferred to a 50 kDa ultrafiltration tube and centrifuged (800 g, 10 min). Sterile PBS (pH 7.4) solution was added to the inner tube of the ultrafiltration tube, mixed evenly, and centrifuged (800 g, 10 min). This washing operation was repeated twice. Finally, the antibody was concentrated to a total volume of 100 μL with PBS (pH 7.4) to obtain an antibody solution with a reduced concentration of 0.096 μg / μL (i.e., a solution containing thiol-aCD3 and thiol-aCD28).

[0173] Grafting of antibody: 17 μL of a 28% (mass concentration) solution of Locked Polyrotaxane (Locked PR synthesized in Example 2, dissolved in Tris-HCl with pH 8.8) was placed in a 96-well polytetrafluoroethylene well plate, and 3 μL of the above-reduced antibody solution was added. It was sonicated for 5 min to mix evenly. It was reacted at 4 °C for 5 hours to obtain an antibody-polyrotaxane solution.

[0174] b. Preparation of Locked Antibody-Polyrotaxane Hydrogel Locked Ab-PR hydr. (abbreviation Locked PR hydr.)

[0175] 1.0 mg of bis-thiol-terminated polyethylene glycol (SH-PEG-SH) (Mw = 5k) was dissolved in 10.0 μL of Tris-HCl (pH 8.8) solution, and then added to the above antibody-polyrotaxane solution, stirred evenly, and reacted at 4 °C for 12 hours to obtain a polyrotaxane gel modified with an antibody. The gel was soaked in ice-cold PBS for 1 hour, and the PBS was discarded. This operation was repeated 4 times to remove unreacted antibodies, polyrotaxanes, and thiol polyethylene glycol. The schematic diagram of the network structure of the obtained Locked PR hydrogel (Locked PR hydr.) is as Figure 2 shown, and the physical object is as Figure 6 shown.

[0176] Example 6 Preparation of Polyrotaxane Hydrogel PR hydr.

[0177] a. Preparation of Antibody-Polyrotaxane Ab-PR

[0178] Reduction of Antibody: The following operations are carried out in a sterile environment. Take 3.0 mg of tris(2-hydroxyethyl)phosphine (TCEP) and dissolve it in 1.0 mL of sterile water to obtain a TCEP aqueous solution with a concentration of 12 mM. Take 120 μL each of aCD3 and aCD28 antibodies (the same as in Example 4) with a concentration of 0.08 μg / μL, mix them evenly with 240 μL of the TCEP aqueous solution, seal, place on a shaker, and react at 4 °C for 2 hours. Transfer the mixed solution to a 50 kDa ultrafiltration tube, centrifuge (800 g, 10 min), add sterile PBS (pH 7.4) solution to the inner tube of the ultrafiltration tube, mix well, and centrifuge (800 g, 10 min). Repeat this washing operation twice. Finally, concentrate the antibody to a total volume of 100 μL with PBS (pH 7.4) to obtain an antibody solution with a reduced concentration of 0.096 μg / μL (i.e., a solution containing thiol-aCD3 and thiol-aCD28).

[0179] Grafting of Antibody: Take 17 μL of a 30% (mass concentration) polyrotaxane solution (PR synthesized in Example 3, dissolved with Tris-HCl at pH 8.8) in a 96-well polytetrafluoroethylene well plate, add 3 μL of the above-reduced antibody solution, mix well, and react at 4 °C for 5 hours to obtain an antibody-polyrotaxane solution.

[0180] b. Preparation of Antibody-Polyrotaxane Hydrogel Ab-PR hydr. (abbreviation: PR hydr.)

[0181] Take 1.0 mg of bis-thiol-terminated polyethylene glycol (SH-PEG-SH) (Mw = 5k) and dissolve it in 10 μL of Tris-HCl (pH 8.8) solution, then add it to the above antibody-polyrotaxane solution, sonicate evenly, and react at 4 °C for 12 hours to obtain a polyrotaxane gel modified with an antibody. Immerse this gel in ice-cold PBS for 1 hour, discard the PBS, and repeat the operation 4 times to remove unreacted antibodies, polyrotaxanes, and thiol polyethylene glycol.

[0182] Example 7 Cytotoxicity Evaluation of PR hydr. and Amplification Effect on CD8 + T Cells

[0183] a. Extraction of Mouse Primary Polyclonal CD8 + T Cells

[0184] Male C57 mice (Vital River Laboratories) around 9 weeks old were euthanized and immersed in 75% disinfected alcohol. Spleen tissues were taken, washed, ground, and red blood cells were lysed to collect spleen lymphocytes. Then, CD8 + T cell negative selection kits (Beaver) were used to isolate CD8 + T cells.

[0185] b. Pretreatment of PR hydr.

[0186] Each piece of PR hydr. hydrogel prepared in Example 4 was gently taken out from the 96-well gel plate and placed in a 48-well plate respectively. 2 mL of PBS solution containing 2% (v / v) penicillin-streptomycin (double antibody) was added and soaked for 3 h. The PBS was aspirated with a pipette, and then the double antibody solution was replaced with 1640 complete medium and soaked at 4°C for 2 h. Then the medium was aspirated again to obtain the pretreated PR hydr.

[0187] c. Amplification of CD8 + T cells

[0188] 200 μL of CD8 6 T cell suspension with a density of 3×10 + cells / mL was added to each piece of the above pretreated PR hydr. hydrogel, gently pipetted and mixed evenly, and placed in an incubator (37°C, 5% CO 2 ) for culture. On the third day, 3-fold volume (3 times the volume) of freshly prepared 1640 complete medium containing 1000 ng / mL of IL-2 was added. From the third day, it was basically possible to see that the medium became significantly yellow (on the premise of excluding any contamination), indicating that the cells were rapidly expanding. On the 5th day, 3-fold volume of freshly prepared 1640 complete medium containing 1000 ng / mL IL-2 was added. The experiment was set up with three replicates.

[0189] d. Collect cells and count

[0190] Cells were collected on the 7th day. 10 μL of cell suspension was taken and diluted to 200 μL with PBS. 1 μL of PI dye was added to label dead cells, and blank control and positive control (the positive control was a commercial reagent Dynabeads (Thermofisher)) (cells were heated at 60°C for 30 min) were set. Counting was performed with a flow cytometer, and the flow rate for each group of tests was set at 30 μL / mL and the time was 3 min. Identify CD8 +T cell subsets: Take a small amount of cell suspension, centrifuge, and sequentially add 4% paraformaldehyde and bovine serum albumin (BSA) blocking solution to fix the cells and block non-specific binding sites. Add 200 μL of PBS to wash, centrifuge, add 200 μL of PBS, add 0.5 μL each of propidium iodide (PI) dye (KeyGen Biotech), PerCP anti-mouse CD44 (Biolegend (USA)), and PE anti-mouse CD62L (Biolegend (USA)), and place at 4°C for 5 h of staining. The experiment is set up with three replicates. The entire amplification operation process is as Figure 8 shown.

[0191] e. Amplification method for the control group

[0192] To evaluate the cytotoxicity and amplification effect of PR hydr., we selected two common amplification methods as the control groups:

[0193] (1) TCP group: Use the conventional method in the laboratory to amplify T cells. The specific steps are as follows: Take a 48-well cell culture plate (with a lysine coating, also known as a tissue culture plate (TCP)), add 100 μL of aCD3 and aCD28 antibody solution with a concentration of 20 μL / mL to the wells, and place in a 37°C incubator (5% CO 2 ) for 2 h of incubation, or place at 4°C for overnight incubation. After incubation, wash the incubation wells 3 times with 200 μL of PBS each time to remove the uncoated antibodies. The subsequent amplification operations are the same as steps b and c of Example 7.

[0194] (2) Dynabeads group (Dyna): Use the clinical conventional CD8 + T cell amplification kit (Dynabeads TM Untouched TM Human CD8 T Cell Kit). The specific steps are as follows: Take the Dynabeads solution, count, take the same number of magnetic beads as the CD8 + T cells to be amplified, wash 2 times with PBS, and then mix it with the CD8 + T cells to be amplified at a ratio of 1:1 in a 48-well plate. The subsequent operations are the same as steps b and c of Example 7.

[0195] f. Cytotoxicity evaluation

[0196] Amplify CD8 according to the method described above +After the T cells, on the seventh day, a small amount of cell suspension was taken into a flow cytometry tube, centrifuged, the supernatant was discarded, and the cells were resuspended in 100 μL of PBS solution. 1 μL of PI solution was added to each tube and incubated at 4 °C for 30 min. Then, the cell viability was measured using a flow cytometer. The experiment was set up with three replicates.

[0197] g. Results

[0198] The results of the cytotoxicity experiments for the PR hydr. group and the TCP group are as Figure 9 shown. It can be seen that after culturing the cells on the hydrogel for 7 days, the cell viability was all between 90% and 95%, indicating that the hydrogel material had almost no cytotoxicity and could also maintain cell viability. The amplification effects of the PR hydr. group, the TCP group, and the Dynabeads group are as Figure 10 shown. It can be seen that it took 7 days for both hydrogels to amplify T cells significantly, and the amplification multiples of the PR Hydr. group and the Locked PR hydr. group were both greater than that of the commercial reagent group, indicating that both hydrogels could be used to efficiently amplify T cells in vitro, and the amplification multiple of the PR hydr. group was greater than that of the Locked PR hydr. group. In addition to evaluating the amplification ability of the two hydrogels from the amplification level, we evaluated the cell function by measuring the cell differentiation. The cell differentiation effects after stimulation in the PR hydr. group and the Dynabeads group are as Figure 11 shown. It can be seen that on the 7th day of amplification, the proportions of effector memory T cells in the T cells of the PR Hydr. group and the Locked PR Hydr. group were 68% and 40% respectively, and the central memory T cells were 3% and 35% respectively. Effector T cells can quickly kill tumor cells and can inhibit tumor growth in a timely manner; while central memory T cells have a long-term immune surveillance function, can stay in the lymph nodes for a long time, and can quickly initiate an immune response to kill tumors when tumor antigens appear again, and can be used to inhibit tumor recurrence. The amplification efficiency of PR hydr. for CD8 + T cells was significantly better than that of the commercial amplification reagent Dynabeads, and the CD8 + T cells activated by PR hydr. could produce more effector T cell subsets.

[0199] Example 8 Cytotoxicity evaluation of Locked PR hydr. and amplification effect on CD8 + T cells

[0200] a. Extract primary CD8 + T cells

[0201] The method was the same as step a in Example 7.

[0202] b. Pretreatment of Locked PR hydr.

[0203] Each Locked PR hydr. hydrogel prepared in Example 5 was gently taken out from the 96-well gel plate, placed in a 48-well plate, and 2 mL of PBS solution containing 2% (v / v) penicillin-streptomycin (double antibody) was added and soaked for 3 h. The PBS was discarded with a pipette, and then the double antibody solution was replaced with 1640 complete culture medium, soaked at 4°C for 2 h, and the culture medium was discarded to obtain the pretreated Locked PR hydr.

[0204] c、Locked PR hydr. Amplification of CD8 + T cells

[0205] Take 200 μL of the solution with a density of 3×10 6 CD8 + The T cell suspension was added to each of the pretreated LockedPR hydrogels, gently blown evenly with a pipette, and placed in an incubator (37°C, 5% CO 2 ) on the third day. On the third day, add 3 times the amount of freshly prepared 1640 complete medium containing 1000 ng / mL IL-2. From the third day, you can basically see that the medium has turned yellow (assuming no contamination), which also shows that the cells are rapidly proliferating. On the fifth day, add 3 times the amount of freshly prepared 1640 complete medium containing 1000 ng / mL IL-2. The experiment was repeated three times.

[0206] d. Collect and count cells

[0207] Same as step d in Example 7.

[0208] e. Evaluation of cytotoxicity and amplification effects

[0209] Same as step f in Example 7.

[0210] Results

[0211] The results of the cytotoxicity test, the expansion effect, and the cell differentiation effect after stimulation of the Locked PR hydr. group are shown in Figures 9 to 11 As shown in the figure, we can see that Locked PR hydr. has an effect on CD8 + The expansion efficiency of T cells was significantly better than that of the commercial expansion reagent Dynabeads, and the CD8 activated by Locked PR hydr. + T cells can generate more memory T cell subsets.

[0212] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A polyrotaxane hydrogel grafted with an antibody, characterized in that: The polyrotaxane hydrogel is at least one of an unlocked polyrotaxane hydrogel PR hydr. and a locked polyrotaxane hydrogel Locked PR hydr.; and is prepared by the following method: S1. Synthesis of Locked PR-ad The adamantane-terminated polyrotaxane was dissolved in an alkaline solution, heated to 40-70°C, epichlorohydrin was added dropwise, stirred for reaction, and after the reaction was completed, dialyzed and freeze-dried to obtain Locked PR-ad; S2. Synthesis of hydroxypropylated polyrotaxane PPG-PR-ad or locked PPG-PR-ad Dissolve the adamantane-terminated polyrotaxane or the locked PR-ad obtained in step S1 in an alkaline solution, add propylene oxide dropwise in an ice bath, stir to react, and after the reaction is completed, dialyze and freeze-dry to obtain PPG-PR-ad or locked PPG-PR-ad; S3. Synthesis of methacrylic acid modified polyrotaxane PR or Locked PR The PPG-PR-ad or Locked PPG-PR-ad obtained in step S2, as well as di-n-butyltin dilaurate and 2,6-di-tert-butyl-p-cresol are dissolved in dimethyl sulfoxide to obtain a polyrotaxane mixed solution; isocyanoethyl methacrylate is dissolved in dimethyl sulfoxide, and then added dropwise to the polyrotaxane mixed solution, and reacted at 37° C. to 60° C. After the reaction is completed, the product is purified and dried to obtain PR or Locked PR; S4. Preparation of antibody-polyrotaxane Ab-PR or Locked Ab-PR Dissolve tri(2-hydroxyethyl)phosphine in water to obtain a TCEP aqueous solution; then add the antibody solution, mix well, and perform a reduction reaction in a sealed container at 4 to 37° C. After the reaction is completed, perform ultrafiltration and centrifugal washing to obtain a reduced thiol-antibody solution; dissolve the PR or Locked PR obtained in step S3 in a buffer solution to obtain a polyrotaxane solution; then evenly mix the reduced thiol-antibody solution and the polyrotaxane solution, and react at 4 to 25° C. to obtain an antibody-polyrotaxane Ab-PR or Locked Ab-PR solution; S5. Preparation of antibody-polyrotaxane hydrogel PR hydr. or Locked PR hydr. Dissolve the dithiol-terminated polyethylene glycol in a buffer solution with a pH of 7.4 to 9.0, and then add it to the antibody-polyrotaxane Ab-PR or Locked Ab-PR solution obtained in step S4, and react at 4 to 25° C. After the reaction is completed, soak the obtained gel in water or a buffer solution to remove unreacted antibodies, polyrotaxanes and thiol polyethylene glycol to obtain an unlocked polyrotaxane hydrogel PR hydr. or a locked polyrotaxane hydrogel Locked PR hydr., that is, the antibody-grafted polyrotaxane hydrogel.

2. The antibody-grafted polyrotaxane hydrogel according to claim 1, characterized in that: The adamantane-terminated polyrotaxane described in steps S1 and S2 is prepared by the following method: (1) Synthesis of terminal carboxyl-grouped polyethylene glycol Dissolve polyethylene glycol in water, add 2,2,6,6-tetramethylpiperidin-1-oxyl free radical and sodium bromide, stir and dissolve, then add sodium hypochlorite solution to adjust the pH of the reaction solution to 10-11, stir and react, add ethanol to quench the reaction after the reaction is completed, then add hydrochloric acid solution to adjust the pH of the reaction solution to <2, extract the reaction solution with dichloromethane, concentrate it by rotary evaporation, and then drip it into ether, collect the white precipitate, and dry it to obtain PEG(COOH)2; (2) Synthesis of pseudo-PR Dissolve the PEG(COOH)2 and α-cyclodextrin obtained in step (1) in hot water at 50-70°C to obtain a PEG(COOH)2 aqueous solution and an α-CD aqueous solution; then add the PEG(COOH)2 aqueous solution dropwise to the α-CD aqueous solution heated to 60±5°C, stir vigorously to react, and freeze-dry after the reaction is completed to obtain pseudo-PR; (3) Synthesis of diamond-terminated polyrotaxane PR-ad The pseudo-PR obtained in step (2), 1-adamantanamine, Carter condensation agent benzotriazole-1-tris(trimethylamino)-trifluorophosphate and N,N-diisopropylethylamine are mixed evenly, and then N,N-dimethylformamide is added dropwise until a sludge is formed, and the mixture is stirred at 4° C. for reaction, and after the reaction is completed, the mixture is washed and freeze-dried to obtain PR-ad; The weight average molecular weight of the polyethylene glycol described in step (1) is 1k to 50k; The molar ratio of polyethylene glycol, 2,2,6,6-tetramethylpiperidin-1-oxyl free radical and sodium bromide in step (1) is 0.3:1.00-3.00:30.0-50.0; The available chlorine content of the sodium hypochlorite solution in step (1) is greater than 5.0%; The mass ratio of α-cyclodextrin to PEG(COOH)2 in step (2) is 4 to 8:1; The concentration of the α-cyclodextrin aqueous solution in step (2) is 2% to 10% by mass; The concentration of the PEG(COOH)2 aqueous solution in step (2) is 1% to 10% by mass; The molar ratio of the pseudorotaxane pseudo-PR, 1-adamantanamine, Carter condensation agent benzotriazole-1-tris(trimethylamino)-trifluorophosphate and N,N-diisopropylethylamine described in step (3) is 1:50-150:5-20:5-20.

3. The antibody-grafted polyrotaxane hydrogel according to claim 2, characterized in that: The weight average molecular weight of the polyethylene glycol described in step (1) is 35K; The molar ratio of polyethylene glycol, 2,2,6,6-tetramethylpiperidin-1-oxyl free radical and sodium bromide in step (1) is 0.3:2.30:36.7; The mass ratio of α-cyclodextrin to PEG(COOH)2 in step (2) is 4:1; The concentration of the α-cyclodextrin aqueous solution in step (2) is 2.4% by mass; The concentration of the PEG(COOH)2 aqueous solution described in step (2) is 1.2% by mass; The molar ratio of the pseudorotaxane pseudo-PR, 1-adamantanamine, Carter condensation agent benzotriazole-1-tris(trimethylamino)-trifluorophosphate and N,N-diisopropylethylamine described in step (3) is 1:100:10:

10.

4. The antibody-grafted polyrotaxane hydrogel according to claim 2, characterized in that: The stirring reaction conditions in step (1) are: stirring the reaction at room temperature for 10 to 20 minutes; The drying conditions described in step (1) are: vacuum drying at 25-80° C. for 5-24 hours; The stirring reaction time in step (2) is 2 to 24 hours; The stirring reaction time in step (3) is 10 to 24 hours; The washing in step (3) is washing with a mixed solution of N,N-dimethylformamide and methanol, methanol, and hot water in sequence; The volume ratio of N,N-dimethylformamide to methanol in the mixed solution of N,N-dimethylformamide and methanol is 1:

1.

5. The antibody-grafted polyrotaxane hydrogel according to claim 1, characterized in that: The mass ratio of the adamantane-terminated polyrotaxane to epichlorohydrin in step S1 is 1:1 to 1.1; In step S1, the concentration of the adamantane-terminated polyrotaxane in the reaction system is 1% to 8% by mass; The alkaline solution described in steps S1 and S2 is an inorganic alkaline solution or an organic alkaline solution; In step S1, the molar ratio of the base in the alkaline solution to the α-cyclodextrin is 20 to 40:1; The molar ratio of epichlorohydrin to α-cyclodextrin in step S1 is 8 to 35:1; The mass ratio of the adamantane-terminated polyrotaxane to propylene oxide in step S2 is 1-1.4:4-5; The mass ratio of the Locked PR-ad to propylene oxide in step S2 is 1-4:4-5; The molar ratio of propylene oxide to α-cyclodextrin in step S2 is 50-150:1; The concentration of the polyrotaxane mixed solution in step S3 is 1% to 4% by mass; The molar ratio of α-cyclodextrin, di-n-butyltin dilaurate, 2,6-di-tert-butyl-p-cresol and isocyanoethyl methacrylate on the PPG-PR-ad or Locked PPG-PR-ad structure in step S3 is 1: 0.01-0.05: 0.003-0.02: 0.1-2; The concentration of the TCEP aqueous solution described in step S4 is 10 to 50 mmol / L; The concentration of the antibody solution in step S4 is 0.08 to 10 μg / μL; The antibodies described in step S4 are aCD3 and aCD28; The concentration of the thiol-antibody solution in step S4 is 0.01 to 10 μg / μL; The mass ratio of the antibody in the thiol-antibody solution to the polyrotaxane in the polyrotaxane solution in step S4 is 1-4:216667; The molecular weight of the bis-thiol terminated polyethylene glycol described in step S5 is 1 to 50K; In step S5, the volume ratio of the dithiol-terminated polyethylene glycol to the antibody-polyrotaxane Ab-PR is 1.5-2.0:1; In step S5, the volume ratio of the dithiol-terminated polyethylene glycol to the antibody-polyrotaxane Locked Ab-PR is 1.5-2.0:

1.

6. The antibody-grafted polyrotaxane hydrogel according to claim 5, characterized in that: In step S1, the concentration of the adamantane-terminated polyrotaxane in the reaction system is 6% by mass; The alkaline solution described in steps S1 and S2 is a sodium hydroxide solution; The concentration of the alkaline solution in steps S1 and S2 is 0.5 to 1.5 mol / L; The amount of the alkaline solution in step S1 is calculated based on 14-16 mL of alkaline solution per gram of PR-ad; In step S1, the molar ratio of the base in the alkaline solution to the α-cyclodextrin is 30:1; The molar ratio of epichlorohydrin to α-cyclodextrin in step S1 is 16:1; The mass ratio of the adamantane-terminated polyrotaxane to propylene oxide in step S2 is 1:5; The mass ratio of Locked PR-ad to propylene oxide described in step S2 is 4:5; The concentration of the polyrotaxane mixed solution in step S3 is 3% to 4% by mass; The molar ratio of α-cyclodextrin, di-n-butyltin dilaurate, 2,6-di-tert-butyl-p-cresol and isocyanoethyl methacrylate on the PPG-PR-ad or Locked PPG-PR-ad structure in step S3 is 1: 0.034-0.043: 0.011-0.02: 0.23-0.45; The concentration of the TCEP aqueous solution described in step S4 is 12 to 20 mmol / L; The concentration of the antibody solution described in step S4 is 0.08 μg / μL; The antibody described in step S4 is an antibody obtained by mixing aCD3 and aCD28 at a mass ratio of 1-2:1-2; The volume ratio of the TCEP aqueous solution to the antibody solution in step S4 is 1:1; The concentration of the thiol-antibody solution described in step S4 is 0.096 μg / μL; The molecular weight of the bis-thiol terminated polyethylene glycol described in step S5 is 5K; In step S5, the volume ratio of the dithiol-terminated polyethylene glycol to the antibody-polyrotaxane Ab-PR is 1.7:1; In step S5, the volume ratio of the dithiol-terminated polyethylene glycol to the antibody-polyrotaxane Locked Ab-PR is 1.7:

1.

7. The antibody-grafted polyrotaxane hydrogel according to claim 1, characterized in that: The temperature of the stirring reaction in step S1 is 50-60°C; The stirring reaction time in step S1 is 6 to 48 hours; The dialysis described in step S1 is performed using a dialysis bag with a molecular weight cut-off of 1000 to 2000 Da; The dialysate used for the dialysis described in steps S1 and S2 is deionized; The stirring reaction time in step S2 is 6 to 24 hours; The dialysis described in step S2 is performed using a dialysis bag with a molecular weight cut-off of 1000 to 2000 Da; The purification in step S3 is performed by organic solvent precipitation, dialysis or extraction; wherein the organic solvent is at least one of acetone, ether and n-hexane; The reaction temperature in step S3 is 40°C to 50°C; The time of the reduction reaction in step S4 is 0.5 to 2 hours; The ultrafiltration in step S4 is performed using a 50 kDa ultrafiltration tube; The washing in step S4 is performed using a PBS buffer solution at pH 7.4; The buffer solution described in step S4 is a Tris-HCl buffer solution with a pH of 8.0 to 9.0; In step S4, the reaction time of the thiol-antibody solution and the polyrotaxane solution is 2 to 24 hours; The buffer solution in step S5 is a Tris-HCl buffer solution of 8.0 to 9.0 or a PBS buffer solution of pH 7.4; The reaction time in step S5 is 1 to 24 hours.

8. The antibody-grafted polyrotaxane hydrogel according to any one of claims 1 to 7 is used to expand CD8 + Application in T cells.

9. An in vitro expansion of CD8 + T cell method, characterized in that: The steps include: I. Pretreatment of Polyrotaxane Hydrogel The antibody-grafted polyrotaxane hydrogel according to any one of claims 1 to 7 is immersed in a PBS buffer solution containing 1% to 5% by volume of penicillin-streptomycin for 1 to 5 hours, then the PBS buffer solution is discarded, a culture medium is added, and the mixture is immersed at 4° C. for 30 minutes to 2 hours, and then the culture medium is discarded to obtain a pretreated polyrotaxane hydrogel; II. Amplification of CD8 + T cells CD8 + The T cell suspension is added to the pretreated polyrotaxane hydrogel obtained in step I, mixed evenly, and cultured at 37°C and 5% CO2. On the third day, 3 to 5 times the volume of culture medium containing 1 to 1000 ng / mL IL-2 is added and cultured. On the fourth or fifth day, 3 to 10 times the volume of culture medium containing 1 to 1000 ng / mL IL-2 is added and cultured. The medium is changed every 2 to 3 days. The cells are collected on the 7th to 14th day.

10. The in vitro expanded CD8 + T cell method, characterized in that: The culture medium described in steps I and II is 1640 complete medium; CD8 as described in step II + The density of T cell suspension was 0.5×10 6 ~5×10 6 Pieces / mL; The concentration of IL-2 in the culture medium described in step II is 1000 ng / mL.

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