Synthesis of setuximab nanospheres and method for coupling setuximab nanospheres with CAR-T cells

Abstract

The invention discloses synthesis of a setuximab nanosphere and a method for coupling the setuximab nanosphere with CAR-T cells, and belongs to the field of biological medicine. According to the invention, NHS-S-S-NHS is used as a cross-linking agent to form a setuximab nanosphere (NGs), and the NGs can be stably attached to the surface of a CAR-T cell through Anti-CD45 and PEG-PLL, and the setuximab nanosphere has the capability of responding to a reductive signal on the surface of the T cell. When the CAR-T cell is excessively activated, an increased reducing environment on the surface of the CAR-T cell triggers the splitting decomposition of the disulfide bond of NHS-S-S-NHS, and the encapsulated setuximab is released to inhibit the cytokine release syndrome (CRS) in situ. According to the invention, the drug can be accurately released as required to relieve the CRS symptom, and the CAR-T cell therapy system has potential clinical utility in the aspect of improving the safety and effectiveness of the CAR-T cell therapy, and has important significance in the aspect of managing unpredictable attack and severity of the CRS.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for synthesizing sildenafil nanospheres and coupling them to CAR-T cells. Background Art

[0002] CAR-T cell therapy has become a powerful tumor immunotherapy strategy for the treatment of many hematological malignancies and even solid tumors. After infusion into the body, CAR-T cells recognize tumor-associated antigens, activate T cells through intracellular signaling domains, stimulate CAR-T cells to proliferate, and secrete cytokines to kill tumor cells. The activation of CAR-T cells is the key to their anti-tumor activity. However, over-activated CAR-T cells will trigger an inflammatory circuit, causing the counter-regulatory homeostasis system to collapse, and lead to a blind increase in cytokine and inflammatory levels in the body, which can easily cause potentially fatal complications in the body, one of which is cytokine release syndrome (CRS). CRS usually occurs in 70% of CAR-T treated patients, and clinical manifestations include high fever, hypotension, hypoxia, vascular leakage, coagulation dysfunction, and severe neurotoxicity. These adverse reactions limit the widespread application of CAR-T cell therapy in patients, and also prompt people to work hard to improve the timely diagnosis and management of CRS and propose a series of prevention and treatment strategies.

[0003] At present, the main therapeutic drugs for CRS are tocilizumab, glucocorticoids, and siltuximab. The goal is to avoid harmful toxicity as much as possible while maximizing the anti-tumor effect of cell therapy. However, this series of drugs is generally administered systemically, which cannot avoid the occurrence of systemic toxic reactions, such as hepatotoxicity, respiratory tract infections, etc. Although the National Cancer Institute of the United States has developed a severity grading scheme for CRS and proposed intervention strategies, it is still necessary to administer drugs only after the CRS reaction is monitored. Therefore, the difficult-to-grasp treatment window has become the biggest challenge in the clinical treatment of CRS. If treatment is given before the onset of CRS symptoms, it will cause a disorder in the body's normal physiological levels. If intervention is performed after the onset of CRS symptoms, permanent damage has already been caused to the patient. Therefore, it is crucial to supply drug treatment in a timely manner at the right time and place. Summary of the invention

[0004] The purpose of the present invention is to provide a method for synthesizing Siltuximab nanospheres and coupling them to CAR-T cells in response to the urgent need for clinical management of the unpredictable onset and severity of CRS. The method selects the clinically approved therapeutic agent Siltuximab, and uses NHS-SS-NHS as a cross-linking agent to form Siltuximab nanospheres (NGs). NGs can stably attach to the surface of CAR-T cells and have the ability to respond to reductive signals on the surface of T cells. When CAR-T cells are overactivated, the increased reducing environment on the surface of CAR-T cells triggers the cleavage of disulfide bonds in the NHS-SS-NHS cross-linker, releasing the encapsulated Siltuximab to inhibit cytokine release syndrome (CRS) in situ. The present invention can achieve precise release of drugs when needed to alleviate CRS-related symptoms, has potential clinical utility in improving the safety and effectiveness of CAR-T cell therapy, and is of great significance in managing the unpredictable onset and severity of CRS.

[0005] The present invention also aims to provide a sildenafil nanosphere, a CAR-T cell with sildenafil nanosphere coupled to its surface, and the pharmaceutical application of the sildenafil nanosphere or the CAR-T cell with sildenafil nanosphere coupled to its surface.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for synthesizing siltuximab nanospheres comprises the following steps: cross-linking siltuximab using NHS-SS-NHS as a cross-linking agent to form siltuximab nanospheres. The cross-linking agent NHS-SS-NHS can respond to a reducing environment, triggering the cleavage of the disulfide bond of the cross-linking agent under the reducing environment to release the encapsulated siltuximab.

[0008] Furthermore, the synthesis method of the siltuximab nanospheres comprises the following steps: stirring a solution containing NHS-SS-NHS and siltuximab to obtain siltuximab nanospheres. The method may further comprise the steps of centrifugation and purification.

[0009] Furthermore, the synthesis method of the siltuximab nanospheres comprises the following steps: dissolving NHS-SS-NHS in dimethyl sulfoxide; dissolving siltuximab in PBS buffer; mixing and stirring the NHS-SS-NHS and siltuximab solution to form siltuximab nanospheres; purifying the siltuximab nanospheres by ultrafiltration centrifugation and dispersing them in PBS.

[0010] A sildenafil nanosphere is obtained by the above synthesis method.

[0011] A method for coupling CAR-T cells with Siltuximab nanospheres comprises the following steps: modifying or adsorbing at least one of Anti-CD45 and polyethylene glycol-b-polylysine (PEG-PLL) onto Siltuximab nanospheres, and then incubating Siltuximab nanospheres with CAR-T cells to couple Siltuximab nanospheres to the surface of CAR-T cells. Anti-CD45 modified Siltuximab nanospheres (NGs) can achieve non-covalent binding of NGs to CAR-T cells, and maintaining NGs on the surface of CAR-T cells significantly prolongs the retention time of NGs in vivo, while maintaining the anti-tumor activity of CAR-T cells. Ethylene glycol-b-polylysine (PEG-PLL) is adsorbed on the surface of NGs, so that NGs carry positive charges, which can promote electrostatic binding between NGs and CAR-T cell membranes.

[0012] Furthermore, the method for coupling CAR-T cells with sildenafil nanospheres comprises the following steps: stirring a solution containing Anti-CD45, PEG-PLL, and sildenafil nanospheres to obtain sildenafil nanospheres modified with PEG-PLL and Anti-CD45; and then incubating the sildenafil nanospheres modified with PEG-PLL and Anti-CD45 with CAR-T cells to couple the sildenafil nanospheres to the surface of CAR-T cells.

[0013] Furthermore, the method for coupling CAR-T cells with Siltuximab nanospheres comprises the following steps: adding Anti-CD45 and PEG-PLL to the Siltuximab nanosphere solution successively or together for stirring, so that Anti-CD45 and PEG-PLL are modified or adsorbed on the Siltuximab nanospheres, and centrifuging and purifying to obtain PEG-PLL and Anti-CD45 modified Siltuximab nanospheres; adding the modified Siltuximab nanospheres to a solution containing CAR-T cells for incubation, so that the Siltuximab nanospheres are coupled to the surface of the CAR-T cells; and centrifuging and collecting to obtain CAR-T cells with surface-coupled Siltuximab nanospheres.

[0014] A CAR-T cell with siltuximab nanospheres coupled to its surface is obtained by the above method. The siltuximab nanospheres are coupled to the surface of the CAR-T cells without being internalized, and the nanospheres are lysed in response to the reducing properties of the CAR-T cell surface to release the loaded siltuximab, thereby treating cytokine release syndrome caused by excessive activation of CAR-T cells.

[0015] The use of the above-mentioned sildenafil nanospheres or CAR-T cells with sildenafil nanospheres coupled to their surfaces in the preparation of drugs.

[0016] A drug comprising the above-mentioned Sildenafil nanospheres or CAR-T cells with Sildenafil nanospheres coupled to their surfaces.

[0017] The indications of the drug include cytokine release syndrome (CRS) caused by excessive activation of CAR-T cells.

[0018] The synthesis of the siltuximab nanospheres provided by the present invention and the method for coupling them to CAR-T cells, as well as the schematic diagram of the release of siltuximab are shown in the following figure. Figure 1 As shown, the method is that siltuximab forms NGs through NHS-SS-NHS cross-linking, and non-covalently binds to CAR-T cells through anti-CD45, so that NGs can be stably attached to the surface of CAR-T cells without being endocytosed, while maintaining the anti-tumor activity of CAR-T cells. And NGs have the ability to respond to reductive signals on the surface of T cells. When CAR-T cells are overactivated, the increased reducing environment on the surface of CAR-T cells triggers the cleavage of disulfide bonds in the NHS-SS-NHS cross-linker, releasing the encapsulated siltuximab to inhibit cytokine release syndrome (CRS) in situ.

[0019] Beneficial effects of the present invention: The present invention can achieve precise release of drugs when needed to relieve CRS-related symptoms, which is of great significance in managing the unpredictable onset and severity of CRS. The present invention can improve the safety of CAR-T cell therapy and enable CAR-T cell therapy to be more widely used. The siltuximab used in the present invention is a clinical drug, and the method of preparing it into nanospheres coupled to the surface of CAR-T cells is simple and convenient for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the synthesis of the siltuximab nanospheres of the present invention and their coupling to CAR-T cells and the release of siltuximab.

[0021] Figure 2 This is a transmission electron microscopy image of Siltoxin nanospheres (NGs).

[0022] Figure 3 It is a CAR-T cell surface reduction assay.

[0023] Figure 4 It is the siltuximab NGs that release siltuximab in response to a reducing environment.

[0024] Figure 5 is the IL-6 content in the serum of mice on the first and seventh days of treatment.

[0025] Figure 6 It is the body temperature difference and weight change of mice during 7 days of treatment.

[0026] Figure 7 is the survival rate of mice within 7 days of treatment. DETAILED DESCRIPTION

[0027] The various aspects taught by the present invention are further described below through the following non-limiting examples. It should be understood that these examples are only some preferred technical solutions, and the scope of protection requested by the present invention is not limited to the following examples.

[0028] Example 1 Preparation of Sildenafil Nanospheres (NGs)

[0029] Preparation method: The preparation process of siltuximab NGs is as follows Figure 1 As shown, NHS-SS-NHS was selected as a cross-linking agent to cross-link siltuximab to form nanospheres (NGs). Specifically, the following steps were included: NHS-SS-NHS (93.5 μg, 0.214 μmol) was dissolved in dimethyl sulfoxide (DMSO, 9.35 μL), siltuximab (1320 μg, 0.0143 μmol) was dissolved in PBS buffer (pH 7.4, 132 μL), NHS-SS-NHS and siltuximab solution were stirred at a constant speed for 30 minutes at 25°C to form NGs, and NGs were purified by ultrafiltration centrifugation and dispersed in PBS (pH 7.4, 1200 μL) at a concentration of 0.35 μg / μL.

[0030] Characterization and performance: According to transmission electron microscopy, the siltuximab NGs prepared in this example have typical spherical structural characteristics, with an average size of about 82.4 nm ( Figure 2 ).

[0031] Example 2 Siltuximab NGs coupled to CAR-T cells

[0032] The sutoxib NGs prepared in Example 1 are coupled to the surface of CAR-T cells to form CAR-T@NGs. Anti-CD45 is selected to modify NGs to achieve non-covalent binding between NGs and CAR-T cells (Wuhan Boruida Biotechnology Co., Ltd.), and maintaining them on the surface of CAR-T cells significantly prolongs the retention time of NGs in vivo while maintaining the anti-tumor activity of CAR-T cells. Polyethylene glycol-b-polylysine (PEG-PLL) is selected to be adsorbed on the surface of NGs, so that NGs carry a positive charge and promote electrostatic binding between NGs and CAR-T cell membranes. Specifically comprising the following steps: Anti-CD45 (215 μg, 0.0014 μmol) is added to the NGs solution (1200 μL, 0.35 μg / μL) prepared above. The mixed solution was stirred at 25°C for 30 minutes, and PEG-PLL (4.36 μg, 0.0014 μmol) was added to the mixed solution (43.6 μL, 0.1 μg / μL) and stirred for 30 minutes to obtain PEG-PLL and Anti-CD45 modified NGs (1 μg / μL). Subsequently, CAR-T cells (1.0×10 7 ) were incubated with NGs (95 μL, 1 μg / μL) at 37°C for 1 hour. Finally, the CAR-T cells with surface-coupled NGs were collected by centrifugation at 800 g for 5 minutes.

[0033] Example 3 Performance evaluation of siltuximab NGs releasing siltuximab in response to a reducing environment

[0034] CAR-T cell surface reduction assay. Specifically, the following steps were included: using the WST-1 assay kit to assay the CAR-T cell surface reduction. The experiment set up an initial group and an activation group, where the initial group was a group of initial CAR-T cells at 1×10 6 The cells were suspended in Hank's balanced salt solution (HBSS) at a concentration of 1×10 cells / mL. 6 ) and IL-7 (1 ng / mL), anti-CD3 / CD28 (1×10 6 Beads / mL) were incubated at 37°C for 24 hours. The cells were washed and resuspended in HBSS and incubated at 37°C for 1 hour, and then the WST-1 reagent mixture (10 μL) was added to the CAR-T cell suspension (200 μL). The absorbance of WST-1 formazan at 450 nm was measured by a microplate reader. Figure 3 As shown, the surface reductivity of activated CAR-T cells increased 6.4 times compared with that of initial CAR T cells.

[0035] Siltuximab NGs released Siltuximab in response to a reducing environment. A reducing environment was created by adding glutathione to the solution. The performance of releasing Siltuximab was evaluated by the following steps: Siltuximab NGs (0.35 μg / mL) were dispersed in PBS without (PBS group) and with GSH (5 mM, GSH group) at 4°C. After 48 hours of co-incubation, the external solution was collected by ultrafiltration centrifugation and the content of Siltuximab was determined by ELISA kit. Figure 4 As shown, compared with the control group, the amount of siltuximab released by siltuximab NGs in the presence of GSH was significantly higher, 3.5 times that of the PBS group.

[0036] Example 4 Siltuximab NGs release Siltuximab in response to a reducing environment for the treatment of cytokine storm (CRS) in mice

[0037] The CAR-T cell-induced CRS model was constructed. First, Raji cells (3×10 6 ) were injected into mice by intraperitoneal injection. Tumor-bearing mice were obtained 21 days later and randomly divided into 4 groups: Control (I), CRS (II), CRS+NGs (III), and CRS+CAR-T@NGs (IV). All drugs were injected by intraperitoneal injection. The Control group was injected with PBS (100 μL) only, and the CRS group was injected with CD19-targeted CAR-T cells (3×10 6 ), CRS+NGs group: injection of CAR-T cells (3×10 6 ) were injected with NGs (dose: 11 mg / kg) before treatment, and the CRS+CAR-T@NGs group: injected with an equivalent dose of CAR-T@NGs. The symptoms of CRS are increased body temperature, weight loss, and increased interleukin (IL)-6 content in serum. Therefore, the weight, body temperature, IL6 content in serum, and survival rate of mice within seven days of treatment were recorded at fixed times. Figure 5 As shown, the mice in the CRS group showed high levels of IL-6 expression, which was 10.6 times higher than that in the control group (Control group), proving the successful construction of the CRS model. The IL-6 content in the serum of mice after NGs injection (CRS+NGs group) decreased by 23.0% compared with the CRS group, proving that the siltuximab released by NGs in response to the reducing environment maintained its original therapeutic activity. And the IL-6 level in the serum of mice injected with CAR-T@NGs (CRS+CAR-T@NGs group) was significantly reduced after seven days, which was 87.7% lower than that of the CRS group. In addition, CRS also exhibits many obvious symptoms, namely increased body temperature and weight loss. As Figure 6As shown, the CRS+NGs group developed symptoms of high fever and weight loss similar to those of the CRS group mice. As expected, the CRS symptoms of mice in the CAR-T@NGs treatment group were significantly alleviated, and the infusion of CAR-T@NGs reduced the CRS-induced mortality rate (reduced by 60%) ( Figure 7 ). It has been demonstrated that CAR-T@NGs can effectively treat and prevent CRS.

[0038] The above description is only a preferred specific implementation manner of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the scope of protection of the invention.

Claims

1. A method for synthesizing siltuximab nanospheres, characterized in that: The following steps are involved: NHS-SS-NHS was used as a cross-linking agent to cross-link siltuximab to form siltuximab nanospheres.

2. The method for synthesizing sutoxib nanospheres according to claim 1, characterized in that: The following steps are involved: The solution containing NHS-SS-NHS and siltuximab is stirred to obtain siltuximab nanospheres.

3. A siltuximab nanosphere, characterized in that: Obtained by the synthesis method according to claim 1 or 2.

4. A method for coupling CAR-T cells with Sildenafil nanospheres, characterized in that: The following steps are involved: At least one of Anti-CD45 and polyethylene glycol-b-polylysine is modified or adsorbed onto the Siltuximab nanospheres, and then the Siltuximab nanospheres are incubated with CAR-T cells to couple the Siltuximab nanospheres to the surface of the CAR-T cells.

5. The method for coupling CAR-T cells with Sildenafil nanospheres according to claim 4, characterized in that: The method comprises the following steps: stirring a solution containing Anti-CD45, polyethylene glycol-b-polylysine and Siltuximab nanospheres to obtain Siltuximab nanospheres modified with polyethylene glycol-b-polylysine and Anti-CD45; and then incubating the modified Siltuximab nanospheres with CAR-T cells to couple the Siltuximab nanospheres to the surface of the CAR-T cells.

6. The method for coupling CAR-T cells with Sildenafil nanospheres according to claim 4, characterized in that: The following steps are involved: Adding Anti-CD45 and polyethylene glycol-b-polylysine to the Siltuximab nanosphere solution successively or together, stirring, and centrifuging and purifying to obtain polyethylene glycol-b-polylysine and Anti-CD45 modified Siltuximab nanospheres; adding the modified Siltuximab nanospheres to a solution containing CAR-T cells for incubation; CAR-T cells with surface-coupled Sildenafil nanospheres were obtained by centrifugation.

7. A CAR-T cell with surface-coupled Siltuximab nanospheres, characterized in that: Obtained by the method according to any one of claims 4 to 6.

8. Use of the sildenafil nanospheres according to claim 3 or the CAR-T cells with surface-coupled sildenafil nanospheres according to claim 7 in the preparation of drugs.

9. A drug, characterized in that: A CAR-T cell comprising the sildenafil nanospheres according to claim 3 or the surface-coupled sildenafil nanospheres according to claim 7.

10. The use according to claim 8 or the medicine according to claim 9, characterized in that: The indications of the drug include cytokine release syndrome.

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