CN115887685B

CN115887685B - Antibody macromolecule conjugate and preparation method and application thereof

Info

Publication number: CN115887685B
Application number: CN202211458515.7A
Authority: CN
Inventors: 梁兴杰; 吴雁; 宫宁强; 李宪磊
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2022-11-21
Filing date: 2022-11-21
Publication date: 2024-02-09
Anticipated expiration: 2042-11-21
Also published as: CN115887685A

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses an antibody macromolecule conjugate and a preparation method and application thereof. The method for preparing the conjugate comprises the following steps: (1) Preparing a copolymer which is poly (N-isopropylacrylamide-co-methacrylic acid); (2) chemically modifying the carboxyl groups of the copolymer; (3) Coupling the copolymer chemically adjusted in step (2) with an antibody. The conjugate has the characteristics of temperature sensitivity and injectability, and can effectively adsorb and reduce the level of inflammatory factors IL-6 in the body after being injected into the body, thereby avoiding undesired body reaction during CAR-T treatment and relieving relevant symptoms.

Description

Antibody macromolecule conjugate and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to an antibody macromolecule conjugate and a preparation method and application thereof.

Background

Currently, some of the pharmacokinetic behavior of immunotherapy or therapeutic approaches still have drawbacks.

For example, immunotherapy has been used as an emerging means for the prevention and treatment of tumors in recent years. Especially for the migrated or diffused tumor, the specific recognition and killing characteristics greatly reduce the toxic and side effects in the treatment process. Cell therapies, typified by CAR-T (chimeric antigen receptor T cell) therapy, have shown great potential in the treatment of hematological diseases such as acute leukemia. At the same time, CAR-T cell therapy also has great potential in the field of lymphomas. CAR-T therapies targeted at the CD19 position are being advanced clinically at great force, exhibiting great potential.

However, CAR-T cells, while killing tumors, interact with other immune cells, stimulating monocytes or macrophages to release a large amount of inflammatory factors, producing a cytokine storm, and producing a series of life-threatening symptoms such as hypotension, high fever, vascular leakage, etc., which severely can lead to organ failure and lethality, we call this phenomenon cytokine release syndrome. In order to alleviate the associated symptoms, some hormonal drugs such as glucocorticoids are used to alleviate or inhibit CAR-T induced cytokine storms. However, these hormonal drugs can also cause very serious damage to the immune system while inhibiting cytokine storms.

Current research evidence suggests that the inflammatory factor interleukin-6 plays an important role in CAR-T induced cytokine storm. Thus, the use of interleukin-6 blocking antibody drugs, including antibodies to interleukin-6 such as cetuximab (Siltuximab) and antibodies to interleukin-6 receptor such as tolizumab (Tocilizumab), has been used clinically to control and manage CAR-T induced cytokine storms and their associated symptoms. However, these blocking antibodies face the following problems and challenges: (1) Injection of these blocking antibodies into the systemic circulatory system is prone to cause toxic side effects such as hyperuricemia, respiratory tract infections, and hepatotoxicity. (2) Clinical studies have shown that tolizumab does not effectively inhibit lethal neurotoxicity, even studies have shown that tolizumab blocks interleukin-6 and its receptor binding, thus resulting in increased free interleukin-6 in the blood and consequently increased interleukin-6 entering the brain, but at the same time, because tolizumab cannot cross the blood brain barrier, neurotoxicity is exacerbated to some extent. (3) More importantly, CAR-T induced cytokine storm management requires the integration of multiple non-specific indicators to determine whether it occurs, when and to what extent there is a serious uncertainty. Because these antibodies are rapidly cleared to the outside of the body, premature administration reduces the inhibitory effect. At the same time, the interleukin-6 plays an important role in maintaining normal physiological activities, so that the interference generated by the interleukin-6 in the body can also influence the normal physiological activities. It will not have therapeutic effects on the cytokine storm after its administration. Even if the administration can be carried out in the storm window period, the rapid metabolism characteristic of the administration greatly reduces the acting time. Therefore, it is urgent to improve the pharmacokinetic behavior thereof or to find more suitable therapeutic methods.

Disclosure of Invention

The present invention is directed generally to a conjugate that is effective in reducing the level of antibody-binding substances (e.g., interleukin-6), thereby avoiding the generation of undesirable bodily reactions (e.g., cytokine storms) and alleviating the associated symptoms.

The specific technical scheme of the invention is as follows:

the present invention provides a method of preparing a conjugate comprising:

(1) Preparing a copolymer which is poly (N-isopropylacrylamide-co-methacrylic acid);

(2) Chemically adjusting the carboxyl groups of the copolymer;

(3) Coupling the copolymer chemically adjusted in step (2) with an antibody.

The conjugate consists of specific temperature-sensitive hydrogel (copolymer) and antibody through chemical coupling, and has the characteristics of temperature sensitivity and injectability, and can be quickly converted into a solid gel state in a room temperature environment and a solid gel state in a body temperature environment, so that the conjugate can be conveniently injected into subcutaneous sites and fixed at the injection sites, and the antibody coupled on the conjugate is fixed at the subcutaneous sites through a specific hydrogel skeleton, so that the pharmacokinetic action form of the conjugate is converted, and the free antibody is blocked and converted into in-situ adsorption, thereby avoiding the generation of undesired body reaction during antibody treatment.

The conjugate of the invention also has temperature reversible property, and can be converted into a sol of liquid from solid gel by a cooling method such as ice compress and the like, and then removed by using a syringe.

The conjugate has porous and adsorption characteristics similar to those of sponge, and can adsorb substances capable of binding antibodies raised in interstitial fluid and blood circulation in situ in real time by chemically coupling specific antibodies, thereby avoiding undesirable related symptoms. For example, the conjugates can act as "cytokine sponges" to reduce serum and brain concentrations of other inflammatory-related factors and to alleviate cytokine storm-related symptoms such as hypotension, vascular leakage, and lethality. Moreover, the conjugate did not affect the killing activity of CAR-T, and did not induce inflammation at the injection site. The conjugate may be aspirated by ice compress for final subcutaneous injection. The preparation method is simple and feasible, and can solve the risk of cytokine storm in the CAR-T treatment process.

In the step (1) of the method of the present invention, the preparation of the copolymer is carried out using N-isopropylacrylamide and methacrylic acid as raw materials and azobisisobutyronitrile as an initiator; the mass ratio of the N-isopropyl acrylamide to the methacrylic acid is (35-38): 1, preferably (36-37): 1.

In step (1) of the process of the present invention, the preparation of the copolymer is carried out under anaerobic conditions, the reaction solvent being anhydrous methanol, the reaction temperature being 65-75 ℃, preferably 70 ℃. The reaction time is 6.5 to 7.5 hours, preferably 7 hours.

The temperature-sensitive hydrogel (copolymer) is a specific high-molecular polymer obtained by copolymerization (free radical polymerization) reaction of two monomers. The polymer is formed with a specific skeleton structure, and can provide a specific coupling carrier for the antibody, so that substances capable of being combined with the antibody can be efficiently adsorbed on the conjugate, the administration mode of the antibody is changed, and the defects that the antibody possibly enters the body to cause toxic and side effects and is easy to be rapidly discharged to the outside are effectively avoided.

In particular, the present inventors have found that poly (N-isopropylacrylamide), while also having a temperature sensitivity, has a backbone structure that is detrimental to subsequent attachment to antibodies. Therefore, the invention introduces the methacrylic acid monomer containing carboxyl with specific structure into the polymer skeleton with specific proportion, so that the structure of the obtained polymer carrier can be effectively connected with the antibody, has porous structure, is favorable for adsorbing substances capable of being combined with the antibody, and can well maintain the temperature sensitivity of the whole material.

As a preferred embodiment, the copolymer of the invention is prepared by using a monomer N-isopropylacrylamide in a dosage range of 960-980mg, preferably 970mg; the charging range of the methacrylic acid is 26-27mg, preferably 26.4mg; the volume of the anhydrous methanol of the reaction solvent ranges from 8 mL to 12mL, preferably 10mL; the amount of azobisisobutyronitrile is in the range of 7-7.5mg, preferably 7.325mg.

In the method of the invention, the chemistry is adjusted to adjust the carboxyl groups in the copolymer to maleimide groups for antibody coupling.

In step (2) of the method of the present invention, the chemical modification is performed with N- (2-aminoethyl) maleimide hydrochloride; the molar ratio of methacrylic acid monomer to N- (2-aminoethyl) maleimide hydrochloride in the copolymer is 1: (1-5), preferably 1:2.

In the invention, the copolymer contains a modifiable carboxyl group, wherein a specific amount of carboxyl groups are adjusted to maleimide groups capable of reacting with protein sulfhydryl groups in a specific manner so as to lay a foundation for the subsequent specific coupling with antibodies, so that the formed conjugate can have a specific antibody coupling structure, thereby being beneficial to the exertion of application effects.

In step (2) of the process of the present invention, 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysulfosuccinimide NHS are used as catalysts; the molar ratio of the N- (2-aminoethyl) maleimide hydrochloride to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysulfosuccinimide is 1 (1-1.2): 1-1.2.

Preferably, the molar ratio of methacrylic acid monomer, maleimide hydrochloride, EDC, NHS in the copolymers of the invention ranges from 1:1:1:1 to 1:5:5:5, preferably 1:2:2:2.

The reaction of step (2) according to the invention is carried out in an aqueous environment, which is Phosphate Buffered Saline (PBS), at a pH of 7.2-7.5, preferably at a pH of 7.4.

The reaction of step (2) is carried out at room temperature for a reaction time of from 23 to 25 hours, preferably 24 hours.

In the step (3) of the method, the mass ratio of the antibody to the copolymer chemically adjusted in the step (2) is 1: (53000-120000), preferably 1: (90000-110000), more preferably 1:100000;

and/or the reaction of step (3) is carried out in an aqueous environment of hydroxyethylpiperazine ethylene sulfate (HEPES) buffer, having a pH of 6.4-6.7, preferably a pH of 6.5;

and/or, the antibody is an interleukin-6 specific antibody.

In step (3) of the present invention, the antibody is coupled to the polymer chain of a specific copolymer by click chemistry, thereby forming a conjugate having a specific structure.

As a preferred embodiment, in the step (3) of the present invention, the system of the buffer in the reaction is 8-12mL, preferably 10mL; the feeding mass of the copolymer after chemical adjustment is 80-120mg, preferably 100mg; the mass of the antibody to be fed is 1 to 1.5. Mu.g, preferably 1. Mu.g.

The preparation method of the invention also comprises the step of concentrating the conjugate coupled in the step (3) to obtain the thermosensitive hydrogel, thereby facilitating the subsequent application.

The preparation method is simple and easy to implement, solves the problem of blocking the in vivo rapid metabolism of the antibody-like drug, changes the blocking of the antibody into in-situ adsorption, improves the inhibition efficiency of the antibody, and avoids the undesired side effect.

The invention also provides a conjugate which is prepared by the method.

The specific surface area of the conjugate of the invention is 9.37-14.14m ² /g；

And/or the concentration of the conjugate is 7.5% -15% (w/w), preferably 10%, so that a larger specific surface area can be obtained, and a suitable injectable and body temperature administration temperature-sensitive effect can be achieved.

The conjugate at a concentration of 10% is a sol that is liquid at room temperature and a gel that is solid at body temperature. The phase transition temperature was 31.5 ℃.

The invention further provides the use of the conjugate in the preparation of a product for preventing or treating CAR-T induced cytokine release syndrome.

The conjugate can be injected and fixed to subcutaneous sites in situ, and the inflammatory factor interleukin-6 circulating in interstitial fluid and serum is adsorbed in situ (through in vivo and in vitro experiments, a good adsorption effect is shown, the level of the inflammatory factor interleukin-6 can be effectively reduced), so that the CAR-T induced cytokine storm and related symptoms thereof can be prevented and inhibited, and the safe anti-tumor effect can be ensured.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be mutually combined to obtain the preferred examples of the invention.

The invention has the advantages that:

in the invention, the conjugate (cytokine sponge based on antibody-macromolecule conjugate) changes the administration mode of the antibody (such as interleukin-6), avoids the possible toxic and side effect caused by the fact that the conjugate enters the systemic blood circulation and the defect of being rapidly discharged to the outside, and prolongs the action window period. The conjugate in the invention has good biological safety, and does not negatively influence the anti-tumor effect of the CAR-T cells. At the same time, since the phase transition of the conjugate is reversible, after the treatment is finished, the conjugate can be removed to the outside by using a syringe through an ice compress method. The conjugates are effective in reducing the level of antibody-binding substances (e.g., interleukin-6), in vitro, in vivo, thereby avoiding the generation of undesirable bodily reactions (e.g., cytokine storms) and alleviating the associated symptoms.

The preparation method is simple and easy to implement, can solve the problem of blocking the medication of the antibody, changes the free blocking into the in-situ adsorption, and solves the problem of pharmacokinetics. The obtained conjugate has a specific three-dimensional configuration and a large specific surface area, is favorable for the combination of antibodies, and can better play the effect of adsorbing substances capable of combining the antibodies.

Drawings

FIG. 1 is a scheme for preparing synthetic antibody-polymer conjugates of the present invention;

FIG. 2 is a diagram showing the nuclear magnetic characterization of the polymer copolymer and the maleimido polymer copolymer of the present invention; wherein the information in the dashed box is an enlarged view of the corresponding region in the figure;

FIG. 3 is a photograph of cytokine sponges (10%) based on antibody-macromolecule conjugates prepared in example 3 at various temperatures;

FIG. 4 is a phase transition diagram of the antibody-macromolecule conjugate-based cytokine sponge prepared in example 3;

FIG. 5 is a scanning electron micrograph of an antibody-macromolecule conjugate-based cytokine sponge prepared in example 3;

FIG. 6 is a graph showing the data of the adsorption of inflammatory factor interleukin-6 by cytokine sponge in vitro in experimental example 1;

FIG. 7 is a graph showing the data of interleukin-6 inflammatory factor in serum of mice in different groups of experimental example 2;

FIG. 8 is a graph showing the body weight of mice in different groups of experimental example 2;

FIG. 9 is a graph showing blood pressure of mice in different groups of Experimental example 2;

FIG. 10 shows the results of the test in Experimental example 3.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.

Reagents and sources used;

n-isopropyl acrylamide, available from Sigma-Aldrich under CAS number 2210-25-5. Preserving at 2-8deg.C.

Methacrylic acid, purchased from Sigma-Aldrich under CAS number 9011-14-7.

Azobisisobutyronitrile, available from Sigma-Aldrich under CAS number 78-67-1.

N- (2-aminoethyl) maleimide hydrochloride, available from TCI. CAS number 134272-64-3.

1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC, available from Sigma-Aldrich under CAS number 1892-57-5.

N-hydroxysulfosuccinimide NHS, available from Sigma-Aldrich under CAS number 6066-82-6.

Mouse interleukin-6 antibody, available from Cell Signaling Technology, cat: 12912T.

400MHz nuclear magnetic resonance spectrometer, instrument model AVANCE III HD 400, available from Bruce, U.S.

Scanning electron microscope, instrument model Hitachi-SU8010, available from Hitachi, japan.

Example 1

The embodiment provides a temperature-sensitive polymer copolymer and a preparation method thereof.

1) 970mg of N-isopropylacrylamide, 26.4mg of methacrylic acid and 7.325mg of azobisisobutyronitrile were each weighed and dissolved in 10mL of anhydrous methanol.

2) The mixture was placed in a three-necked flask, air in the reaction system was evacuated at room temperature, and then nitrogen was purged for 30 minutes to block oxygen.

3) The reaction was warmed to 70℃and stirred uniformly using a magnet, and the whole process was kept under reflux by condensation and reacted for 7 hours. The reactants changed from a liquid to a transparent solid.

4) The reaction was cooled naturally to room temperature while maintaining a nitrogen atmosphere.

5) Spin-steaming for 1 hour, and removing excessive methanol in the system as much as possible.

6) Adding a proper amount of deionized water into the three-neck flask, swelling to synthesize hydrogel, taking out, dialyzing for three days, and removing unreacted micromolecules and impurities. Freeze-drying for preservation.

The synthetic route of the high molecular copolymer is shown in fig. 1 (upper diagram). The characterization of the macromolecular copolymer nuclear magnetism (deuterated water test) is shown in fig. 2 (upper graph).

Example 2

In this example, the polymer copolymer obtained in example 1 was further chemically modified to have a maleimide group, thereby obtaining a maleinized polymer copolymer.

1) 215mg of the polymer copolymer, 17.6mg of N- (2-aminoethyl) maleimide hydrochloride, 29mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and 17.2mg of N-hydroxysulfosuccinimide NHS were weighed, respectively.

2) Then, it was dissolved in PBS and reacted at room temperature with stirring for 24 hours.

3) The reacted samples were collected, dialyzed for three days to remove excess N- (2-aminoethyl) maleimide hydrochloride, EDC and NHS, and finally lyophilized samples for material characterization.

The synthetic route of the obtained maleimido high molecular copolymer is shown in FIG. 1 (middle graph). The characterization of the maleinized polymer nuclear magnetism (deuterated water test) is shown in fig. 2 (lower panel).

Example 3

This example provides an antibody-macromolecule conjugate (cytokine sponge) and a method of making the same.

1) First, 100mg of the maleinized polymer copolymer prepared in example 2 was dissolved in 10mL of HEPES buffer system at ph=6.5. Then, 1. Mu.g of interleukin-6 antibody was added to the reaction system. And (3) reacting for 24 hours at room temperature to obtain the antibody-macromolecule conjugate.

2) The antibody-macromolecule conjugate was concentrated to 1mL using an ultrafiltration tube. A cytokine sponge with 10% temperature sensitivity was obtained. Storing at 4deg.C.

The preparation route of the antibody-macromolecule conjugate is shown in figure 1 (lower panel);

the state of the obtained cytokine sponge based on the antibody-polymer conjugate at different temperatures is shown in fig. 3, wherein the room temperature is 25 ℃, and the body temperature is 37 ℃. From these, it is understood that the cytokine sponge obtained in this example has an injectable effect, i.e., it is in a liquid state at room temperature of 25℃and in a solid state at 37 ℃.

The phase transition temperature of the cytokine sponge based on the antibody-macromolecule conjugate is shown in figure 4, and the microscopic morphology (electron scanning photograph) is shown in figure 5.BET specific surface area of 14.14m ² /g。

Experimental example 1

In vitro adsorption experiments were performed on the cytokine sponge prepared in example 3.

To the bottom of the 96-well plate, 5. Mu.l of the cytokine sponge prepared in example 3 was added dropwise, and then placed in a 37-degree cell incubator. After it had turned into a solid gel, 100. Mu.l of a PBS solution containing 600pg/mL interleukin-6 was slowly added thereto. After standing at 37℃for 2 hours, the supernatant was assayed for the concentration of interleukin-6 remaining using an Elisa kit, thereby calculating the amount of cytokine sponge adsorbed. The test results are shown in FIG. 6. Wherein the control is an IL-6 solution without cytokine sponge.

Experimental example 2

In this experimental example, an adsorption experiment in mice was performed on the cytokine sponge prepared in example 3, and the effect of alleviating the symptoms associated with cytokine storm was examined. Group 3 biological replicates. The specific method comprises the following steps:

will be 3X 10 ⁶ Individual tumor cells (Raji) were injected into the peritoneal cavity of healthy SCID-beige (6-8 weeks, female) mice. Three weeks later, one experimental group (cytokine sponge group) was obtained by injecting 100uL of the cytokine sponge prepared in example 3 into the subcutaneous lower right side of tumor-loaded SCID-beige mice, and the other experimental group (IL-6 antibody group) was obtained by intravenous injection of an equivalent dose of IL-6 antibody (100 ng), and an untreated group was additionally used as a control. Immediately following intraperitoneal injection of 30X 10 mice in each group ⁶ CAR-T cells (inducing inflammatory storms). Mice were monitored for body weight and blood pressure changes. Two days after CAR-T cell injection, mouse serum was collected and the concentrations of inflammatory factor IL-6 in the different groups of mouse serum were tested using the Elisa kit.

The inflammatory factor interleukin-6 concentration in the serum of different groups of mice is shown in FIG. 7; body weights of different groups of mice are shown in fig. 8; blood pressure of the mice in the different groups is shown in FIG. 9.

From the results, the cytokine sponge prepared in example 3 can effectively adsorb interleukin 6 in vivo, and relieve symptoms (control weight and blood pressure drop) related to cytokine storm.

Experimental example 3

The experimental example tests the effect of the cytokine sponge prepared in example 3 on the anti-tumor effect of CAR-T cells. The experimental steps are as follows:

will be 3X 10 ⁶ Individual tumor cells (Raji, luciferase expression) were injected into the peritoneal cavity of healthy SCID-beige (6-8 weeks, female) mice. Three weeks later, one experimental group (cytokine sponge group) was obtained by injecting 100uL of the cytokine sponge prepared in example 3 into the subcutaneous lower right side of tumor-loaded SCID-beige mice, and the other experimental group (IL-6 antibody group) was obtained by intravenous injection of an equivalent dose of IL-6 antibody (100 ng), and an untreated group was additionally used as a control. Immediately following intraperitoneal injection of 30X 10 mice in each group ⁶ CAR-T cells (inducing inflammatory storms). At various time points after CAR-T treatment, the changes in signal of Raji cells in different groups of mice were monitored using a small animal fluorescence imager by intraperitoneal injection of D-fluorescein sodium salt (reacting with Raji-expressed luciferase to generate a fluorescent signal, the stronger the signal representing the more Raji cells in the body, the better the antitumor effect). The effect of cytokine sponge on the anti-tumor effect of CAR-T was evaluated and the results are shown in figure 10.

Comparative example 1

The present comparative example provides an antibody-polymer conjugate and a method for preparing the same, comprising:

(1) The preparation of the polymer copolymer was carried out in the same manner as in example 1, except that: the raw material ratio of the N-isopropyl acrylamide and the methacrylic acid is changed, and the specific steps are as follows: 970mg of N-isopropylacrylamide, 36mg of methacrylic acid and 7.325mg of azobisisobutyronitrile were each weighed and dissolved in 10mL of anhydrous methanol, respectively, with the remaining steps unchanged.

(2) The preparation of the maleimided high molecular copolymer was carried out according to the method of example 2, except that the amounts of the respective substances were as follows: 215mg of a polymer copolymer, 10mg of N- (2-aminoethyl) maleimide hydrochloride, 29mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and 17.2mg of N-hydroxysulfosuccinimide NHS.

(3) The preparation of the antibody-macromolecule conjugate was performed according to the method of example 3.

Finally, the temperature sensitivity of the hydrogel obtained in the comparative example is reduced, and the hydrogel with various concentrations cannot form solid under the state of body temperature.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A method of preparing a conjugate comprising:

(2) Chemically adjusting the carboxyl groups of the copolymer;

(3) Coupling the copolymer chemically adjusted in step (2) with an antibody;

in the step (1), N-isopropyl acrylamide and methacrylic acid are used as raw materials, and azobisisobutyronitrile is used as an initiator to prepare the copolymer; the mass ratio of the N-isopropyl acrylamide to the methacrylic acid is (35-38): 1, a step of;

in step (2), the chemical modification is performed with N- (2-aminoethyl) maleimide hydrochloride; the molar ratio of methacrylic acid monomer to N- (2-aminoethyl) maleimide hydrochloride in the copolymer is 1: (1-5).

2. The method according to claim 1, wherein in the step (1), the preparation of the copolymer is performed under an oxygen-free condition, the reaction solvent is anhydrous methanol, and the reaction temperature is 65 ℃ to 75 ℃.

3. The method according to claim 2, wherein in the step (1), the reaction temperature is 70 ℃.

4. A method according to any one of claims 1 to 3, wherein in step (2) the molar ratio of methacrylic acid monomer to N- (2-aminoethyl) maleimide hydrochloride in the copolymer is 1:2.

5. The method according to claim 4, wherein in the step (2), 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide are used as catalysts; the molar ratio of the N- (2-amino ethyl) maleimide hydrochloride to the 1-ethyl- (3-dimethyl amino propyl) carbodiimide to the N-hydroxy thiosuccinimide is 1 (1-1.2): 1-1.2;

and/or the reaction of the step (2) is carried out in an aqueous phase environment, wherein the aqueous phase environment is phosphate buffer solution, and the pH value is 7.2-7.5.

6. The method of claim 5, wherein in step (2), the aqueous environment has a pH of 7.4.

7. The method of any one of claims 1-3, 5-6, wherein in step (3), the mass ratio of the antibody to the chemically modified copolymer of step (2) is 1: (53000-120000);

and/or the reaction of the step (3) is carried out in an aqueous phase environment, wherein the aqueous phase environment is a hydroxyethyl piperazine ethylsulfuric acid buffer solution, and the pH value is 6.4-6.7;

and/or, the antibody is an interleukin-6 antibody.

8. The method of claim 4, wherein in step (3), the mass ratio of the antibody to the chemically modified copolymer of step (2) is 1: (53000-120000);

and/or, the antibody is an interleukin-6 antibody.

9. The method of claim 7, wherein in step (3), the pH of the aqueous environment is 6.5.

10. The method of claim 8, wherein in step (3), the pH of the aqueous environment is 6.5.

11. A conjugate prepared by the method of any one of claims 1 to 10.

12. The conjugate according to claim 11, wherein the specific surface area of the conjugate is 9.37-14.14m ² /g；

And/or the concentration of the conjugate is 7.5% -15% (w/w).

13. The conjugate of claim 12, wherein the concentration of the conjugate is 10% (w/w).

14. Use of a conjugate according to any one of claims 11-13 for the preparation of a product for preventing or treating CAR-T induced cytokine release syndrome.