Application of hydrogel cultured CAR-M / CAR-T in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention relates to the technical field of immunotherapy, in particular to a method for preparing CAR-M and / or CAR-T with enhanced cell killing ability. The method comprises the steps that CAR-M and / or CAR-T are / is cultured on hydrogel, so that the CAR-M and / or CAR-T with the enhanced cell killing capacity are / is obtained, the hydrogel is gelatin-oxidized sodium alginate cross-linked hydrogel, and the oxidation degree is 50%; the material has the energy storage modulus of 1953 to 1120Pa, the loss modulus of 112.0 to 42.81 Pa, the loss coefficient of 0.06945 to 0.03730 and the stress relaxation coefficient of 2915 to 2019s. The CAR-M cell therapy is introduced into pulmonary fibrosis treatment for the first time, and hydrogel with optimal parameters is combined, so that the treatment efficiency of CAR-M / CAR-T cells is improved, the killing ability is improved by at least four times, and pulmonary tissue fibrosis is effectively relieved.

Description

Technical Field

[0001] This application relates to the field of immunotherapy technology, specifically to a hydrogel for preparing chimeric antigen receptor macrophage CAR-M and / or chimeric antigen receptor T cell CAR-T with enhanced cell-killing ability, a method for preparing CAR-M and / or CAR-T using the same, its use in preparing drugs for treating pulmonary fibrosis, and its use in improving the cell-killing ability of CAR-M. Background Technology

[0002] Pulmonary fibrosis is a difficult-to-treat interstitial lung disease. In the early stages of the disease, fibrosis can be reduced through medication, smoking cessation, or avoiding harmful environmental factors. Currently, only two drugs are approved for the treatment of pulmonary fibrosis: pirfenidone and nintedanib. Both drugs have adverse reactions. Pirfenidone may cause photosensitivity, nausea and vomiting, and abnormal liver function, while nintedanib may cause diarrhea, nausea and vomiting.

[0003] In recent years, immunotherapy (such as CAR-T therapy) has made significant progress in cancer treatment and is gradually being applied to the treatment of lung diseases. However, on the one hand, CAR-T cells cannot infiltrate solid tumors, and their therapeutic effect on lung diseases remains questionable; on the other hand, some studies have shown that CAR-T cell therapy for lung cancer patients still has cytotoxic side effects. Macrophages are important cells in the immune system. They can engulf pathogens and cancer cells, and present antigens to activate T cells, often allowing them to enter solid tumors. Therefore, chimeric antigen receptor macrophages (CAR-M) are considered to be highly promising therapeutic cells. Studies have found that different biomaterial substrates can induce macrophage function in different ways, serving as effective tools for regulating macrophages.

[0004] Therefore, there is an urgent need for a method to prepare chimeric antigen receptor macrophages (CAR-M) with enhanced cell-killing ability, and to effectively treat pulmonary fibrosis by targeting fibroblasts in the lungs with modified CAR-M. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems in the related art.

[0006] Therefore, an embodiment of the first aspect of the present invention provides a method for preparing chimeric antigen receptor macrophages and / or chimeric antigen receptor T cells with enhanced cell-killing ability, comprising:

[0007] Chimeric antigen receptor macrophages (CAR-M) and / or chimeric antigen receptor T cells (CAR-T) are cultured on a hydrogel to obtain CAR-M and / or CAR-T cells with enhanced cell-killing ability, wherein the hydrogel has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel with an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s.

[0008] In some embodiments, the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1317±185.4 Pa, loss modulus of 66.34±13.81 Pa, loss coefficient of 0.05±0.0061, and stress relaxation coefficient of 2589±368.7 s.

[0009] In some embodiments, the energy storage modulus is 1317 Pa, the loss modulus is 66.34 Pa, the loss coefficient is 0.05, and the stress relaxation coefficient is 2589 s.

[0010] In some embodiments, the CAR-M and / or CAR-T have the following characteristics: reduced membrane tension; CAR protein uniformly distributed on the membrane surface; and increased number of CAR protein monomers and dimers.

[0011] In some embodiments, the CAR-M and / or CAR-T are FAP5-CAR M and / or anti-CD19 CAR-T.

[0012] In some embodiments, the gelatin-sodium oxidized alginate crosslinked hydrogel is prepared by the following steps:

[0013] Sodium alginate was oxidized using sodium periodate to obtain oxidized sodium alginate with an oxidation degree of 50%.

[0014] The oxidized sodium alginate and gelatin were mixed to obtain a pre-crosslinked hydrogel; and

[0015] The initially cross-linked hydrogel is immersed in a calcium ion solution for secondary cross-linking to obtain the gelatin-sodium oxidized alginate cross-linked hydrogel.

[0016] A second aspect of the present invention provides a hydrogel for preparing CAR-M and / or CAR-T cells with enhanced cell-killing ability, wherein the hydrogel has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel has an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s.

[0017] In some embodiments, the hydrogel is prepared by the following steps: oxidizing sodium alginate with sodium periodate to obtain oxidized sodium alginate with an oxidation degree of 50%; mixing the oxidized sodium alginate with gelatin to obtain a pre-crosslinked hydrogel; and impregnating the pre-crosslinked hydrogel in a calcium ion solution for secondary crosslinking to obtain the gelatin-oxidized sodium alginate crosslinked hydrogel.

[0018] In some embodiments, the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1317±185.4 Pa, loss modulus of 66.34±13.81 Pa, loss coefficient of 0.05±0.0061, and stress relaxation coefficient of 2589±368.7 s.

[0019] In some embodiments, the energy storage modulus is 1317 Pa, the loss modulus is 66.34 Pa, the loss coefficient is 0.05, and the stress relaxation coefficient is 2589 s.

[0020] An embodiment of the third aspect of the present invention provides the use of hydrogel-cultured CAR-M and / or CAR-T in the preparation of a medicament for treating pulmonary fibrosis, wherein the hydrogel has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel having an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s; the hydrogel-cultured CAR-M and / or CAR-T have the following characteristics: reduced membrane tension; uniformly distributed CAR protein on the membrane surface; and increased number of CAR protein monomers and dimers.

[0021] An embodiment of the fourth aspect of the present invention provides a kit for preparing a drug for treating pulmonary fibrosis, comprising: a hydrogel, wherein the hydrogel is used to culture CAR-M and / or CAR-T and has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel having an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730 and stress relaxation coefficient of 2915-2019 s; and CAR-M and / or CAR-T, wherein the CAR-M and / or CAR-T, after being cultured in the hydrogel, are used to treat pulmonary fibrosis.

[0022] A fifth aspect of the present invention provides the use of a hydrogel in enhancing the cell-killing ability of CAR-M and / or CAR-T cells, characterized in that the hydrogel has the following features: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel having an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s; wherein the CAR-M and / or CAR-T cells cultured on the hydrogel have the following characteristics: reduced membrane tension; uniformly distributed CAR protein on the membrane surface; and increased number of CAR protein monomers and dimers.

[0023] In some embodiments, the CAR-M and / or CAR-T are FAP5-CAR M and / or anti-CD19 CAR-T.

[0024] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0025] (1) CAR-M cell therapy was introduced into the treatment of a mouse model of pulmonary fibrosis for the first time. It was found that the therapy could not only effectively clear activated fibroblasts in the lung tissue, but also significantly reduce the degree of pulmonary fibrosis, showing an effective relief effect on pulmonary fibrosis in mice, and opening up a new avenue for the treatment of pulmonary fibrosis.

[0026] (2) For the first time, a hydrogel with preferred parameters is provided that can enhance the function of CAR-M and CAR-T cells, which further enhances the therapeutic efficacy of CAR-M cells and increases the killing ability by at least 4 times. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the CAR framework structure used to prepare chimeric antigen receptor macrophages (CAR-M) in the embodiments of the present invention.

[0028] Figure 2 Is it transfection? Figure 1 Flow cytometry plot of CAR protein expression in CAR-Jurkat cells within the CAR framework shown.

[0029] Figure 3 Is it transfection? Figure 1 The flow cytometry diagram of CD69 protein expression in CAR-Jurkat cells co-cultured with target cells within the CAR framework shown.

[0030] Figure 4 Is it transfection? Figure 1 Flow cytometry plot of CAR protein expression in CAR-THP cells within the CAR framework shown.

[0031] Figure 5 Is it transfection? Figure 1 Flow cytometry plot of CAR protein expression in CAR-BMDM cells within the CAR framework shown.

[0032] Figure 6 This is a schematic diagram of the preparation process of gelatin-sodium oxidized alginate crosslinked hydrogel according to an embodiment of the present invention.

[0033] Figure 7 This is a graph showing the detection results of viscoelastic parameters of hydrogels with different oxidation levels according to an embodiment of the present invention.

[0034] Figure 8 This is a graph showing the test results of the stress relaxation properties of hydrogels with different oxidation levels according to an embodiment of the present invention.

[0035] Figure 9 The diagram shows the cytotoxic function detection results of two CAR-M (Gel-CAR-M) cultured in hydrogel according to an embodiment of the present invention.

[0036] Figure 10 A schematic diagram illustrating the statistical levels of Gel-CAR-M cytokine expression in an embodiment of the present invention is shown.

[0037] Figure 11 The graph shows the results of Gel-CAR-M membrane tension detection according to an embodiment of the present invention.

[0038] Figure 12 This diagram illustrates the fluorescence microscopy imaging and statistical representation of the CAR protein on the membrane surface of Gel-CAR-M according to an embodiment of the present invention.

[0039] Figure 13A schematic diagram showing the results of non-reducing CAR protein immunoblotting and statistical analysis of the membrane surface CAR protein of Gel-CAR-M according to an embodiment of the present invention.

[0040] Figure 14 This is a schematic diagram of the protocol for cell therapy of pulmonary fibrosis using Gel-CAR-M according to an embodiment of the present invention.

[0041] Figure 15 This is a statistical graph showing the weight change of mice treated with pulmonary fibrosis using Gel-CAR-M according to an embodiment of the present invention.

[0042] Figure 16 This is a schematic diagram showing the statistical content of hydroxyproline in mouse lung tissue after pulmonary fibrosis cell therapy using Gel-CAR-M according to an embodiment of the present invention.

[0043] Figure 17 These are three staining results of mouse lung tissue treated with pulmonary fibrosis using Gel-CAR-M according to an embodiment of the present invention.

[0044] Figure 18 The diagram shows the results of the killing ability detection of Gel-CAR-T cells cultured in hydrogels with different oxidation degrees and the schematic diagram of their gene expression levels according to an embodiment of the present invention. Detailed Implementation

[0045] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0046] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0047] Pulmonary fibrosis is a difficult-to-treat interstitial lung disease. In the early stages of the disease, fibrosis can be reduced through medication, smoking cessation, or avoiding harmful environmental factors. Currently, only two drugs are approved for the treatment of pulmonary fibrosis: pirfenidone and nintedanib. Both drugs have adverse reactions. Pirfenidone may cause photosensitivity, nausea and vomiting, and abnormal liver function, while nintedanib may cause diarrhea, nausea and vomiting.

[0048] In recent years, immunotherapy (such as CAR-T therapy) has made significant progress in cancer treatment and is increasingly being applied to the treatment of lung diseases. However, on the one hand, CAR-T cells cannot infiltrate solid tumors; on the other hand, some studies have shown that CAR-T cell therapy for lung cancer patients still has cytotoxic side effects. Macrophages are important cells in the immune system; they can engulf pathogens and cancer cells, and present antigens to activate T cells, often allowing them to enter solid tumors. Therefore, chimeric antigen receptor macrophages (CAR-M) are considered to be highly promising therapeutic cells.

[0049] Macrophage polarization plays a crucial role in pulmonary fibrosis. Related technologies have been studied for the treatment of cardiac fibrosis using CAR-M therapy. Other research has explored the large-scale differentiation of macrophages from embryonic stem cells for the treatment of liver fibrosis in mice.

[0050] Studies have found that different biomaterial substrates can induce macrophage function in different ways. Biomaterial hydrogels and physiological extracellular matrices are not linear elastic materials, but rather viscoelastic materials, a property more complex than simple hardness. Common biomaterial hydrogels, such as type I collagen gel, reconstituted basement membrane matrix, and fibrin gel, all exhibit viscoelasticity, meaning that under external force, their strain behavior displays characteristics of both elastic and viscous materials, capable of both permanent and elastic deformation. Stress relaxation is a phenomenon unique to viscoelastic materials, referring to the gradual decrease in internal stress over time under constant temperature and deformation. Biomaterials can regulate macrophage activation by altering their physical properties, changing cell membrane tension, and influencing the function and distribution of all membrane proteins. Therefore, it is believed that different biomaterial substrates can induce macrophage function in different ways and can serve as effective tools for macrophage regulation.

[0051] However, no studies have yet provided suitable biomaterial hydrogels for inducing CAR-M and treating pulmonary fibrosis with CAR-M cultured in hydrogels.

[0052] Therefore, an embodiment of the first aspect of the present invention provides a method for preparing chimeric antigen receptor macrophages and / or chimeric antigen receptor T cells with enhanced cell-killing ability, comprising:

[0053] Chimeric antigen receptor macrophages (CAR-M) and / or chimeric antigen receptor T cells (CAR-T) are cultured on a hydrogel to obtain CAR-M and / or CAR-T cells with enhanced cell-killing ability, wherein the hydrogel has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel with an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s.

[0054] In some embodiments, the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1317±185.4 Pa, loss modulus of 66.34±13.81 Pa, loss coefficient of 0.05±0.0061, and stress relaxation coefficient of 2589±368.7 s.

[0055] In some embodiments, the energy storage modulus is 1317 Pa, the loss modulus is 66.34 Pa, the loss coefficient is 0.05, and the stress relaxation coefficient is 2589 s.

[0056] In some embodiments, the CAR-M and / or CAR-T have the following characteristics: reduced membrane tension; CAR protein uniformly distributed on the membrane surface; and increased number of CAR protein monomers and dimers.

[0057] In some embodiments, the CAR-M and / or CAR-T are FAP5-CAR M and / or anti-CD19 CAR-T.

[0058] In some embodiments, the gelatin-sodium oxidized alginate crosslinked hydrogel is prepared by the following steps:

[0059] Sodium alginate was oxidized using sodium periodate to obtain oxidized sodium alginate with an oxidation degree of 50%.

[0060] The oxidized sodium alginate and gelatin were mixed to obtain a pre-crosslinked hydrogel; and

[0061] The initially cross-linked hydrogel is immersed in a calcium ion solution for secondary cross-linking to obtain the gelatin-sodium oxidized alginate cross-linked hydrogel.

[0062] In some specific embodiments, the preparation process of gelatin-sodium oxidized alginate crosslinked hydrogel is as follows: Figure 6As shown, sodium alginate was oxidized using sodium periodate to obtain oxidized sodium alginate with abundant aldehyde groups. The degree of oxidation of sodium alginate was controlled in this step, preparing oxidized sodium alginate with oxidation degrees of 20%, 50%, and 95%. Then, the oxidized sodium alginate and gelatin were thoroughly mixed. The aldehyde groups in the oxidized sodium alginate reacted with the amino groups in the gelatin to form Schiff base covalent bonds. Finally, the hydrogel, initially cross-linked by Schiff bases, was impregnated into a solution containing calcium ions. The calcium ions chelated the hydroxyl groups of sodium alginate to form calcium ion chelate bonds, completing the secondary cross-linking. Through the two cross-linking reactions, Schiff base covalent bonds and calcium ion chelate ionic bonds were formed respectively. Furthermore, the proportions of the two types of bonds formed by the oxidized sodium alginate with different oxidation degrees were also different. In hydrogels with lower oxidation levels, the Schiff base content is lower and the calcium ion chelation degree is higher, resulting in a faster stress relaxation rate. In contrast, in hydrogels with higher oxidation levels, the Schiff base content is higher and the calcium ion chelation degree is lower, resulting in a slower stress relaxation rate.

[0063] A second aspect of the present invention provides a hydrogel for preparing CAR-M and / or CAR-T cells with enhanced cell-killing ability, wherein the hydrogel has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel has an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s.

[0064] It should be noted that the above ranges are the maximum and minimum values ​​of the viscoelastic parameters measured through the embodiments in this article.

[0065] In some embodiments, the hydrogel is prepared by the following steps: oxidizing sodium alginate with sodium periodate to obtain oxidized sodium alginate with an oxidation degree of 50%; mixing the oxidized sodium alginate with gelatin to obtain a pre-crosslinked hydrogel; and impregnating the pre-crosslinked hydrogel in a calcium ion solution for secondary crosslinking to obtain the gelatin-oxidized sodium alginate crosslinked hydrogel.

[0066] In some embodiments, the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1317±185.4 Pa, loss modulus of 66.34±13.81 Pa, loss coefficient of 0.05±0.0061, and stress relaxation coefficient of 2589±368.7 s. It should be noted that the above ranges are the average values ​​± SD of the viscoelastic parameters measured through the embodiments described herein.

[0067] In some embodiments, the storage modulus is 1317 Pa, the loss modulus is 66.34 Pa, the loss coefficient is 0.05, and the stress relaxation coefficient is 2589 s. It should be noted that the above ranges are average values ​​of the viscoelastic parameters measured through the embodiments described herein.

[0068] An embodiment of the third aspect of the present invention provides the use of hydrogel-cultured CAR-M and / or CAR-T in the preparation of a medicament for treating pulmonary fibrosis, wherein the hydrogel has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel having an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s; the hydrogel-cultured CAR-M and / or CAR-T have the following characteristics: reduced membrane tension; uniformly distributed CAR protein on the membrane surface; and increased number of CAR protein monomers and dimers.

[0069] An embodiment of the fourth aspect of the present invention provides a kit for preparing a drug for treating pulmonary fibrosis, comprising: a hydrogel, wherein the hydrogel is used to culture CAR-M and has the following characteristics: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel having an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730 and stress relaxation coefficient of 2915-2019 s; and CAR-M and / or CAR-T, wherein the CAR-M and / or CAR-T, after being cultured in the hydrogel, are used to treat pulmonary fibrosis.

[0070] A fifth aspect of the present invention provides the use of a hydrogel in enhancing the cell-killing ability of CAR-M and / or CAR-T cells, characterized in that the hydrogel has the following features: the hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel, the gelatin-sodium oxidized alginate crosslinked hydrogel having an oxidation degree of 50%; the viscoelastic parameters of the gelatin-sodium oxidized alginate crosslinked hydrogel are as follows: storage modulus of 1953-1120 Pa, loss modulus of 112.0-42.81 Pa, loss coefficient of 0.06945-0.03730, and stress relaxation coefficient of 2915-2019 s; wherein the CAR-M and / or CAR-T cells cultured on the hydrogel have the following characteristics: reduced membrane tension; uniformly distributed CAR protein on the membrane surface; and increased number of CAR protein monomers and dimers.

[0071] In some embodiments, the CAR-M and / or CAR-T are FAP5-CAR M and / or anti-CD19 CAR-T.

[0072] Example

[0073] laboratory animals

[0074] Wild-type male C57BL / 6J mice (CD45.1 and CD45.2 subtypes) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After purchase, the mice were transported to the barrier enclosure of the Tsinghua University Laboratory Animal Center according to the prescribed procedures. The mice were housed in a sterile barrier with a 12-hour dark-light cycle and were fed JAX-standard mouse feed (Synerbio, catalog number: SZS9126) and sterile water. All animal experiments conducted in this work were performed after approval by the Tsinghua University Laboratory Animal Use and Management Committee.

[0075] Chemical reagents

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The product numbers of the reagents used in the examples are shown in Table 1 below.

[0077] Table 1

[0078]

[0079]

[0080] Data statistics

[0081] Statistical analyses in these examples were performed using GraphPad Prism 6.0 software. Data are presented as mean ± standard deviation; other methods will be described in the text if applicable. The Anderson-Darling test, D'Agostino-Pearson test, Shapiro-Wilk test, and Kolmogorov-Smirnov test were used to check whether the sample data conformed to a normal distribution. For significance analysis of two groups, if the data conformed to a normal distribution, a t-test was used; otherwise, the Mann-Whitney test was used. For significance analysis of multiple groups, if the data conformed to a normal distribution, one-way ANOVA and Tukey multiple comparisons were used; otherwise, the Kruskal-Wallis test was used. Other methods will be described in the text if applicable. All data included at least three biological replicates. Specific p-values ​​are indicated in the figures, where p < 0.05 was considered statistically significant.

[0082] Example 1: Construction and Screening of CAR-M

[0083] In this embodiment, three different CARs targeting the FAP protein—single-stranded variable fragment (scFv)73.3, FAP5, and MO36—were collected, and three corresponding lentiviral plasmids were generated. CAR proteins were expressed in the Jurkat cell line using these three lentiviral plasmids, and co-cultured with primary activated fibroblasts of mouse lung as target cells to screen for effective CARs. Then, CAR-M cell lines and primary cells expressing the CAR protein were obtained using the lentiviral plasmids corresponding to the effective CARs.

[0084] 1.1 Plasmid Construction

[0085] (1) The nucleic acid sequences of scFv 73.3, FAP5 and MO36 were directly synthesized, and BstBI and MluI restriction sites were introduced at their 5' and 3' ends.

[0086] (2) Insert the scFv into the CAR frame to provide, for example Figure 1 The CAR plasmids shown include anti-FAPCAR and control plasmids, and the lentiviral vector plasmid promoter is FE1α.

[0087] (3) Using the above-mentioned plasmids and three packaging helper plasmids pMD2.G, pRRE, and pRSV-Rev, and using the 293T cell line as packaging cells, the plasmids were added in a ratio of 4:2:2:1. For a 10μm cell culture dish, the total amount of plasmid added was 30μg. That is, 60ul of Lipo293TM and 30μg of plasmid were added to 1ml of Opti-MEM medium, mixed evenly, and allowed to stand at room temperature for 15 minutes. Then, the mixture was slowly added dropwise to a freshly cultured dish after changing the medium, and cultured for another 12 hours.

[0088] (4) Replace with 10ml of fresh culture medium. After 48 hours, collect the supernatant and store it at 4℃. Replace with 10ml of fresh culture medium again. After 24 hours, collect the supernatant and store it at 4℃. Combine the supernatants collected twice and centrifuge at 500×g for 10 minutes to obtain the virus supernatant. Then filter the virus supernatant using a 0.22μm filter membrane. Subsequently, centrifuge the virus supernatant at 19500rpm for 2.5 hours. Resuspend the virus particles using Opti-MEM and aliquot and store at -80℃.

[0089] This step yielded lentiviral plasmids containing scFv 73.3, FAP5, and MO36, as well as a positive control.

[0090] 1.2 Screening for anti-FAP scFv expression on membrane

[0091] (1) The virus was added to 1640 completely fresh cell culture medium (1640 basal medium + 10% FBS + 1% P / S double antibody) to infect Jurkat cells at a virus titer ratio of MOI=10. After waiting for 48 hours, three types of CAR-Jurkat cells were obtained.

[0092] (2) After harvesting the cells, enrich the cells and place them on ice, then resuspend them in flow cytometry buffer (PBS solution containing 2% FBS).

[0093] (3) Add primary anti-goat anti-mouse Fab antibody, incubate on ice for 1 hour, then dilute the staining solution with flow cytometry buffer and centrifuge at 500×g for 5 minutes to wash away the primary antibody.

[0094] (4) Resuspend the cells in an appropriate amount of flow cytometry buffer, add the fluorescently labeled secondary antibody, incubate on ice in the dark for 1 hour, then dilute the staining solution with flow cytometry buffer and centrifuge at 500×g for 5 minutes to wash away the secondary antibody.

[0095] (5) Cells were resuspended in an appropriate amount of flow cytometry buffer and analyzed to assess the expression levels of various CARs on the cell membrane.

[0096] The results are as follows Figure 2As shown, the CAR of 73.3 scFv failed to be effectively expressed on the Jurkat cell membrane, while the CARs composed of FAP5 and MO36 scFv were successfully expressed on the surface of the Jurkat cell membrane. Under the same dose of lentivirus infection, FAP5-CAR showed a membrane uptake efficiency of 21.9% on Jurkat cells, and MO36-CAR showed a membrane uptake efficiency of 18.1% on Jurkat cells.

[0097] 1.3 Effective anti-FAP scFv screening

[0098] After CAR-T cells are activated by contact with target cells, they can highly express CD69 protein molecules on their cell membranes. This example utilizes the contact between CAR-T cells and target cells to screen for effective anti-FAP scFv.

[0099] (1) Take lungs from 8-10 week old C57BL / 6J male mice and digest them completely into a single-cell suspension using an enzymatic method (0.1% type I collagenase + 0.1% type IV collagenase + 0.01% hyaluronidase + 0.01% DNase I, dissolved in DMEM basal medium).

[0100] (2) Centrifuge to remove the digestion solution, and culture overnight in DMEM complete medium (i.e., DMEM basal medium + 10% FBS + 1% P / S double antibiotic mixture) to allow fibroblasts to gradually adhere to the wall, and then remove the medium to obtain mouse primary lung fibroblasts.

[0101] (3) Primary fibroblasts were activated in vitro using DMEM complete cell culture medium containing 10 ng / ml TGF-β factor for three days to obtain activated fibroblasts with high expression of FAP protein.

[0102] (4) Activated fibroblasts were used as target cells and co-cultured with FAP5-Jurkat cells and MO36-Jurkat cells at a 1:1 ratio for 12 hours.

[0103] (5) Collect CAR-Jurkat cells and use flow cytometry to detect the expression level of CD69 protein in them.

[0104] The results are as follows Figure 3The image shows flow cytometry results of CD69 protein expression after co-culturing wild-type Jurkat cells, FAP5-Jurkat cells, and MO36-Jurkat cells with primary activated lung fibroblasts. The first row shows the negative control cells without co-culture, the second row shows the cells after co-culture, and the third row shows the positive control cells stimulated by PMA. The arrows on the horizontal axis represent the fluorescence intensity of EGFRt tag protein staining, and the arrows on the vertical axis represent the fluorescence intensity of CD69 protein staining. The numbers in the boxes represent the percentage of positive cell populations; each group underwent the same staining procedure.

[0105] In this experiment, the negative control consisted of unco-cultured cells. It was observed that cells not co-cultured, whether Jurkat or CAR-Jurkat, were in a resting, inactive state, with nearly zero CD69-positive cells. The positive control consisted of cells activated using conditioned medium containing 100 ng / ml phorbol 12-myristate 13-acetate (PMA), with over 90% of these cells being CD69-positive. In this experiment, the CD69-positive percentage in Jurkat cells co-cultured with primary activated lung fibroblasts was 2.97%, while in CAR-Jurkat cells it was 21.8% and 54.2%, respectively. This indicates that CAR-Jurkat can be activated by target cells and upregulate CD69 protein expression. These results demonstrate that the CAR composed of FAP5 and MO36 scFv can not only be normally expressed in Jurkat cells but also effectively activated upon contact with activated fibroblasts, validating the effectiveness of these two CARs.

[0106] 1.4 Preparation of CAR-M cell lines

[0107] After validating the effectiveness of CAR on the Jurkat cell line, this embodiment uses the same lentivirus to infect the THP1 cell line (human myeloid leukemia mononuclear cell line) to construct CAR-M cells.

[0108] (1) THP cells were seeded in 1640 complete medium (i.e., RPMI 1640 basal medium + 10% FBS + 1% P / S double antibody-induced medium). After the cells reached an appropriate density, they were infected with control lentivirus and two anti-FAPCAR lentiviruses, respectively.

[0109] (3) After infection, the EGFRt protein was screened and enriched by flow cytometry based on the tag.

[0110] (4) The expression of CAR protein on the membrane of enriched CAR-THP cells was detected.

[0111] (5) The enriched CAR-THP cells were differentiated using 200 ng / ml PMA conditioned medium for 48 hours.

[0112] The results are as follows Figure 4 The results showed that both CARs could be successfully expressed on the surface of THP cells, and high-purity CAR-THP cells with a positive rate of over 90% could be obtained after screening and enrichment for subsequent experiments. Furthermore, detection of CD68 and CD11b macrophage markers in THP cells after differentiation stimulation demonstrated that they could successfully differentiate into typical macrophages under PMA stimulation, providing favorable conditions for subsequent experiments.

[0113] 1.5 Preparation of CAR-M primary cells

[0114] (1) Take the tibia and fibula of 8-12 week old C57BL / 6 male mice and wash them three times each with 75% alcohol and sterile PBS.

[0115] (2) Cut open one end of the tibia and fibula, and centrifuge at 5000 rpm for 5 minutes to collect bone marrow cells from the medullary cavity.

[0116] (3) Use DMEM with 10% FBS and 1% PS as complete cell culture medium. On this basis, use concentrated L929 supernatant as differentiation factor and add it to the cell culture medium at a ratio of 10× as differentiation condition medium.

[0117] (4) Resuspend bone marrow cells in differentiation-conditioning medium. Resuspend bone marrow cells from one bone in 10 ml of DMEM complete cell culture medium. Place the cell suspension in a 10 cm cell culture dish and culture for differentiation in a 37°C cell culture incubator for 7 days to obtain bone marrow-derived mononuclear cells.

[0118] (5) Differentiate bone marrow-derived monocytes into macrophages using conditioned medium containing M-CSF. Differentiation can be completed in seven days.

[0119] (6) On the fifth day of differentiation, the cells were infected with a viral titer of MOI=30. After infection, the cells were cultured for another two days to allow CAR to be fully expressed in the primary macrophages and to allow the macrophages to complete their differentiation.

[0120] (7) Flow cytometry was used to detect the expression of CAR on the surface of primary macrophages, and wild-type BMDM cells were used as controls for box selection and average fluorescence intensity analysis.

[0121] The results are as follows Figure 5As shown, flow cytometry plots of CAR protein expression are displayed for wild-type BMDM cells, control BMDM cells, FAP5-BMDM cells, and MO36-BMDM cells. The arrows on the horizontal axis represent the fluorescence intensity of CAR protein staining. The numbers in the boxes represent the percentage of positive cell populations. The bar chart is a statistical graph of the average fluorescence intensity of CAR protein staining. The same staining procedure was performed on each group.

[0122] Figure 5 The results showed that FAP5-CAR had a positivity rate of 34.2% in BMDM cells, while MO36-CAR had a positivity rate of 24.7%. The mean fluorescence intensity of FAP5-BMDM cells was also significantly higher than that of MO36-BMDM cells, indicating that FAP5-CAR is better expressed in primary macrophages. For primary macrophages, viral infection efficiency is crucial, requiring a cell population with the highest possible positivity rate for various in vitro and in vivo experiments. Therefore, FAP5-CAR was chosen for subsequent experiments.

[0123] Example 2: Hydrogel Preparation and Detection

[0124] In this embodiment, sodium alginate with different oxidation degrees was prepared, and these oxidized sodium alginate were further crosslinked with gelatin to prepare hydrogels required for subsequent experiments. The viscoelasticity and stress relaxation properties of the prepared hydrogels were then tested.

[0125] 2.1 Hydrogel Preparation

[0126] First, weigh 5g of sodium alginate powder and add it to 15ml of anhydrous ethanol. To prepare sodium alginate with oxidation degrees of 20%, 50%, and 95%, weigh 1.0694g, 2.6735g, and 5.0797g of sodium periodate respectively and dissolve them in 15ml of deionized water. Shake thoroughly in the dark to dissolve the sodium alginate. In a dark environment, maintain a temperature of 37°C and magnetically stir the sodium alginate powder. Slowly add the sodium periodate solution dropwise. After the addition is complete, continue heating and stirring the mixture at 37°C for 6 hours. After the oxidation reaction is complete, add 3ml of ethylene glycol to terminate the reaction and continue stirring for 30 minutes. Transfer the mixture to a dialysis bag for dialysis. Continue stirring and dialysis for 3 days to obtain an oxidized sodium alginate solution. Filter the oxidized sodium alginate solution through a 0.22μm filter membrane for sterilization, then freeze and lyophilize it. Seal and store the lyophilized oxidized sodium alginate, weighing it each time for use. Weigh an appropriate amount of gelatin particles and dissolve them in 0.9% sodium chloride solution at 37°C to prepare a 20% gelatin solution. Filter the gelatin solution through a 0.22 μm filter to sterilize it. Weigh an appropriate amount of lyophilized sodium alginate and dissolve oxidized sodium alginate in PBS buffer to prepare a 20% solution. Mix equal volumes of the gelatin solution and the oxidized sodium alginate solution thoroughly, then add the mixture to well plates. Place the plates in a 37°C environment for 24 hours to allow gelation. After removing the plates, drop a layer of 0.1M calcium chloride solution onto the gel surface and allow it to stand at room temperature for 3 hours to form a gel. The prepared hydrogel can be sealed and stored at 4°C.

[0127] 2.2 Testing of the viscoelasticity and stress relaxation properties of hydrogels

[0128] The viscoelasticity of the hydrogel was measured using the mmi CellManipulator optical capture system (mmi Cellmanipulator, MM1 AG, ​​Germany). Polystyrene microspheres (Hugebio, China) with a diameter of 5 μm were embedded on the scaffold surface. Individual microspheres were captured using a 1070 nm, 8 W laser, with the laser moving sinusoidally at a displacement amplitude of 0.5 μm and a frequency of 1 Hz. The viscoelastic characteristics of the scaffold material were evaluated by recording the amplitude-time curves of the microsphere response. For specific measurement methods, please refer to Wu, Y., Liang, H., Luo, A., Li, Y., Li, X., & Li, W., et al. (2023). Gelatin-based 3d biomimetic scaffolds platform potentiates culture of cancerstemcells in esophageal squamous cell carcinoma. Biomaterials, 302.

[0129] The results of viscoelastic parameters of hydrogels with different oxidation degrees are as follows: Figure 7 As shown, n>30 represents multiple data points from 3 or more biologically independent replicates, and the data are presented as mean ± standard deviation. Figure 7 Part (a) shows the initial storage modulus (G') of the hydrogel, representing the elastic part of the viscoelastic properties, which describes the solid-state characteristics of the material. The results show that hydrogels with different oxidation degrees maintain the consistency of the initial storage modulus. Figure 7 Part (b) shows the loss modulus (G”) of the hydrogel. The loss modulus represents the viscous portion of the viscoelastic properties and describes the liquid characteristics of the material. The results show that the loss modulus gradually decreases with increasing oxidation of the hydrogel, indicating that hydrogels have different loss moduli while maintaining a consistent storage modulus. Figure 7 Section (c) shows the loss coefficient (tanθ) of the hydrogel, which is the ratio of the material's loss modulus to its storage modulus. The fact that the three groups of hydrogels have different loss coefficients indicates that they have different viscoelasticities. This result shows that the entire hydrogel system has controllable viscoelastic variations.

[0130] Test of stress relaxation properties of hydrogels with different oxidation degrees, such as Figure 8 As shown, (a) displays the stress relaxation curves of hydrogels with different oxidation states, and (b) displays the apparent half-life τ(1 / 2) of the stress relaxation coefficient of hydrogels with different oxidation states, where n = 5, representing 5 biologically independent replicates. Data are presented as mean ± standard deviation. The results show that hydrogels with different oxidation states exhibit different stress relaxation curves.

[0131] In summary, the results show that the storage modulus of the hydrogel with 20% oxidation is 1346±154.5 Pa, the loss modulus is 174.0±32.85 Pa, the loss coefficient is 0.1304±0.04369, and the stress relaxation coefficient is 994.7±130.1 s. The storage modulus of the hydrogel with 50% oxidation is 1317±185.4 Pa, the loss modulus is 66.34±13.81 Pa, the loss coefficient is 0.05020±0.006084, and the stress relaxation coefficient is 2589±368.7 s. The storage modulus of the hydrogel with 95% oxidation degree is 1392±228.5 Pa, the loss modulus is 12.76±7.129 Pa, the loss coefficient is 0.009710±0.005490, and the stress relaxation coefficient is 4331±528.3 s (all are shown here as average value ± SD).

[0132] The results of subsequent embodiments show that viscoelasticity and stress relaxation coefficient have a significant impact on cell function.

[0133] Example 3: CAR-M cultured in hydrogel and its functional determination

[0134] 3.1 CAR-M cultured in hydrogel

[0135] (1) FAP5-CAR-BMDM and FAP5-CAR-RAW cells (CAR-M cells obtained by transfection of another macrophage RAW264.7 cell line with lentivirus) were prepared using the steps in Examples 1.3 and 1.4.

[0136] (2) CAR-M cells were cultured on hydrogels with oxidation levels of 20%, 50%, and 95% as treatment groups. At the same time, CAR-M cells were induced by ordinary culture dishes, 100 ng / ml LPS factor and 20 ng / ml IL4 factor respectively, and cultured together in a cell culture incubator at 37°C for 24 hours. Then, the CAR-M cells cultured in hydrogel (Gel-CAR-M) and other control group cells were harvested.

[0137] 3.2 Lethality Detection

[0138] The macrophages and target cells (mouse primary lung fibroblasts obtained according to Example 1.3) obtained in 3.1 above were co-cultured at a ratio of 2:1, and their killing ability against target cells was continuously observed over 4 days.

[0139] Adding Aka luciferase substrate to the culture medium of active 3T3Aka cells resulted in strong bioluminescence, while adding the same dose of Aka luciferase substrate to the culture medium of inactive 3T3Aka cells produced no bioluminescence. We used a microplate reader to record the bioluminescence intensity of 3T3Aka cell populations co-cultured with CAR-M cells after killing the cells, and compared it with the bioluminescence intensity of 3T3Aka cell populations cultured alone without killing the cells. The difference was calculated and compared with the bioluminescence intensity of 3T3Aka cell populations cultured alone without killing the cells; this ratio is the killing ratio indicated in the figure. Based on this calculation principle, we statistically analyzed the killing efficiency of CAR-THP cells against target cells.

[0140] Gel-CAR-M lethality test results are as follows: Figure 9 As shown, the results of Gel-CAR-RAW kill function test (n=6) and Gel-CAR-BMDM kill efficiency test (n=3) are presented respectively.

[0141] Macrophages can be polarized into two subtypes, M1 and M2. The classic method involves using LPS to polarize macrophages into M1 and IL-4 to polarize them into M2. Generally, M1 macrophages are pro-inflammatory, producing pro-inflammatory factors, and M1 CAR-M cells should exhibit stronger cytotoxic effects. The results of this embodiment demonstrate that, using LPS-induced M1 macrophages as a control, the CAR-M cells cultured in hydrogel in this embodiment have stronger cytotoxic capabilities compared to LPS-induced CAR-M cells.

[0142] As shown in the figure, CAR-M cells cultured on ordinary cell culture dishes exhibited cytotoxic activity against target cells. CAR-M cells induced by LPS and IL-4 did not show enhanced cytotoxicity, while CAR-M cells cultured on hydrogels showed enhanced cytotoxicity, with the 50% oxidizing hydrogel group exhibiting the strongest cytotoxic activity. In the CAR-BMDM group, the cytotoxic activity of Gel-CAR-BMDM was almost four times higher than that of the other control groups. This was completely unexpected. Subsequent related measurements were all performed using 50% oxidizing hydrogels.

[0143] 3.3 Detection of Cytokine Changes

[0144] (1) CAR-RAW and Gel-CAR-RAW cells were prepared using the steps in Examples 1.3 and 3.1.

[0145] (2) CAR-RAW cells and Gel-CAR-RAW cells were co-cultured with target cells (mouse primary lung fibroblasts obtained according to Example 1.3) at a ratio of 2:1 for 24 hours, and the expression levels of representative inflammatory factors iNOS and IL6 after cell activation were detected.

[0146] The results are as follows Figure 10 As shown, the gene expression levels of the inflammatory cytokines iNOS and IL6 are detected (n=3). Both iNOS and IL6 are representative cytokines secreted after CAR-M activation, and both were significantly elevated in the Gel-CAR-M group. This result indicates that hydrogel culture enables CAR-M to respond more strongly to target cell stimulation.

[0147] 3.4 Membrane tension detection

[0148] (1) CAR-RAW and Gel-CAR-RAW cells were prepared using the steps in Examples 1.3 and 3.1.

[0149] (2) Prepare serum-free DMEM medium and add FliptR fluorescent probe at a concentration of 1 μM.

[0150] (3) Resuspend CAR-RAW and Gel-CAR-RAW cells in 1.5ml EP tubes using culture medium with probes at a density of 1 million cells / ml.

[0151] (4) Prepare a quarter dish, pipette, and pipette tip. Place at 37°C for at least 30 minutes, protect from light, and bring to the FLIM microscope.

[0152] (5) Before loading the sample, add 100 μl to one well of the confocal-specific quartering dish and place it on the sample stage for 5 minutes to allow the cells to settle.

[0153] (6) Excite the fluorescent probe with 488nm excitation light and detect the emitted light with a 600nm filter.

[0154] (7) The membrane tension of each group of cells was measured using the membrane tension analysis module.

[0155] The results are as follows Figure 11 As shown, the membrane tension of Gel-CAR-M cells cultured on hydrogel was significantly lower than that of CAR-M cells, indicating that hydrogel affects cell membrane tension. This may be because the hydrogel transmits signals to the cell through proteins on the membrane, thereby affecting cell function.

[0156] 3.5 Detection of CAR protein distribution on membrane

[0157] (1) CAR protein was fused with mCherry fluorescent protein and CAR-RAW and Gel-CAR-RAW cells were prepared using the steps in Examples 1.3 and 3.1.

[0158] (2) Collect CAR-M and Gel-CAR-M cells and fix the cells with paraformaldehyde.

[0159] (3) CAR protein was labeled with mCherry fluorescent protein, cell membrane was labeled with DiR dye, and cells were observed using a high-resolution fluorescence microscope.

[0160] (4) The aggregation of monomers and dimers of CAR protein on cells was detected at the molecular level using a non-reducing Western blot method.

[0161] Microscopic observation results as follows Figure 12As shown, section (a) illustrates high-resolution imaging and mean fluorescence intensity analysis of macrophages modified with the fused mCherry fluorescent protein CAR. The scale bar in the figure is 5 μm. n = 3 represents images from 3 biologically independent replicates, and data are presented as mean ± standard deviation. Section (b) illustrates total internal reflection fluorescence microscopy imaging of the CAR protein and statistical analysis of its distribution. The scale bar in the figure is 5 μm. n = 10 represents multiple cells from more than 3 biologically independent replicates, and data are presented as mean ± standard deviation.

[0162] from Figure 12 As shown in section (a), CAR proteins formed obvious clusters on the surface of CAR-M cell membranes, while CAR proteins on Gel-CAR-M cell membranes did not form obvious clusters but were uniformly dispersed. ImageJ software analysis revealed that the aggregation degree of CAR proteins on CAR-M cell membranes was significantly higher than that on Gel-CAR-M cells.

[0163] from Figure 12 As can be seen in part (b), more CAR protein clusters can be formed on the CAR-M cell membrane, while fewer CAR protein clusters are formed on the Gel-CAR-M cell membrane, and the area of ​​the clusters is also smaller.

[0164] Western blot results are as follows Figure 13 As shown, (a) part displays the non-reducing immunoblot image of Gel-CAR-M cells, with the size and location of CAR protein monomers and dimers marked; (b) part shows the statistical analysis of the proportion of monomers and dimers in the non-reducing immunoblot. As can be seen from the figure, more CAR protein monomers and dimers appear in the immunoblot lanes of Gel-CAR-M cells. This indicates that hydrogel culture transforms the CAR protein molecules on the cell membrane from their original clustered form to monomeric or dimer forms, increasing the probability of CAR protein contact with target cells and enabling CAR-M cells to respond to target cell stimulation more quickly and strongly.

[0165] In summary, CAR-M cells cultured on hydrogels exhibited enhanced cytotoxicity, with the 50% oxidation hydrogel group showing the strongest cytotoxic activity. In the CAR-BMDM group, the cytotoxic activity of Gel-CAR-BMDM was at least four times higher than that of other control groups. The expression levels of representative inflammatory factors iNOS and IL6 were significantly increased in the Gel-CAR-M group. The cell membrane tension of Gel-CAR-M cells cultured on hydrogels was significantly lower than that of CAR-M cells, and the CAR protein in the Gel-CAR-M cell membrane was more uniformly distributed, containing more CAR protein monomers and dimers.

[0166] In summary, the 50% oxidation degree hydrogel, through its optimized viscoelasticity and oxidation degree, reduces the membrane tension of CAR-M cells cultured on it, promotes the uniform distribution of CAR proteins, increases the number of CAR protein monomers and dimers, and ultimately significantly enhances its killing ability.

[0167] Example 4: Treatment of pulmonary fibrosis using CAR-M cultured in hydrogel

[0168] This embodiment uses, as follows Figure 14 The procedure shown demonstrates CAR-M cell therapy in a mouse model of pulmonary fibrosis.

[0169] (1) Using 10-week-old male C57BL / 6 mice, a 5 mg / ml bleomycin solution was prepared using PBS buffer, and bleomycin was injected into the trachea of ​​anesthetized mice at a dose of 1 μl / g.

[0170] (2) CAR-M cell therapy was administered to mice with pulmonary fibrosis by injecting cells at 7 and 14 days after bleomycin induction. The study included a Vehicle group (PBS), a macrophage therapy group (M), a CAR-M cell therapy group (CAR-M), and a hydrogel-treated CAR-M cell therapy group (Gel-CAR-M). At 7 and 14 days, each cell injection group received 1 × 10⁻⁶ cells via tail vein injection. 6 The mice were injected with one cell per 100 cells, while the PBS group received an equal volume of PBS. Twenty-one days after bleomycin induction, mouse lung samples were collected and fibrotic changes were examined.

[0171] (3) Record the weight of mice within 21 days after bleomycin induction.

[0172] (4) The changes in hydroxyproline content in the lungs of mice treated with cell therapy were determined using a hydroxyproline content kit.

[0173] (4) Using the Masson tricolor reagent kit, hematoxylin-eosin and Sirius red stain, the lungs of mice were stained with Sirius red, hematoxylin-eosin and Masson tricolor according to the conventional staining method to detect the degree of pulmonary fibrosis.

[0174] Mouse weight changes as follows Figure 15 As shown in the figure. At the final weighing of the mice, the weights of the mice in each group were compared: the healthy group had the heaviest weight, the fibrosis group had the lightest weight, and the Gel-CAR-M cell therapy group had significantly higher weights than the other treatment groups and the PBS group.

[0175] The hydroxyproline content in mouse lung tissue is as follows Figure 16As shown, the hydroxyproline content in the lungs of mice in the macrophage injection group did not decrease significantly and remained essentially the same as that in the PBS group. However, the hydroxyproline content in the lungs of mice in the CAR-M and Gel-CAR-M groups decreased compared to the PBS group. In particular, the Gel-CAR-M group showed a significantly lower level than the PBS group. These results indicate that macrophage treatment has a certain degree of improvement on pulmonary fibrosis in mice, mainly due to CAR-M cells and Gel-CAR-M cells, with GelCAR-M showing the best therapeutic effect.

[0176] Mouse lung staining results as follows Figure 17 As shown, (a) shows the results of the Masson trichrome staining and its statistical analysis; (b) shows the results of the hematoxylin-eosin staining and the statistical analysis of the Ashcroft scores; and (c) shows the results of the Sirius red staining and its statistical analysis.

[0177] Masson's trichrome staining results showed that the collagen area in the lungs of the M group, CAR-M group, and Gel-CAR-M group was significantly reduced compared to the PBS group, with the Gel-CAR-M group showing the best therapeutic effect. Hematoxylin-eosin staining results indicated that cell therapy in the CAR-M and Gel-CAR-M groups had significant effects in reducing fibrous tissue deposition, restoring alveolar structure, and alleviating inflammatory responses, with the Gel-CAR-M group showing a lower score. Sirius red staining results also showed a larger collagen area in the lungs of fibrotic mice, and CAR-cell and Gel-CAR-M cell therapy groups showed significant improvement in lung collagen fiber deposition, especially the Gel-CAR-M group, which showed a significant improvement compared to the lungs of fibrotic mice.

[0178] In summary, these results indicate that cell therapy in groups M, CAR-M, and Gel-CAR-M all had a certain impact on pulmonary fibrosis in mice. Among them, CAR-M and Gel-CAR-M cell therapy significantly improved pulmonary fibrosis in mice, mainly by reducing collagen deposition in the lungs, improving lung tissue structure, restoring alveolar structure, and reducing inflammatory cell infiltration. These results demonstrate the effectiveness of CAR-M cell therapy for pulmonary fibrosis in mice and show that Gel-CAR-M cells have better therapeutic effects in practical applications.

[0179] Example 5

[0180] In this embodiment, anti-CD19 CAR-T cells were prepared according to Example 1.4, and anti-CD19 CAR-T cells were cultured using hydrogels with different oxidation states according to Example 3.1. Furthermore, the killing ability of Gel-CAR-T cells was determined according to Example 3.2. In addition, the gene expression levels of CAR-T cell killing-related genes and exhaustion-related genes in Gel-CAR-T cells were also detected.

[0181] Test results as follows Figure 18 As shown.

[0182] Figure 18 Part A shows the relative killing ability of anti-CD19 CAR-T cells cultured in well plates and hydrogels, with the killing ability of CAR-T cells cultured in well plates as a control. Hydrogels with 20% and 50% oxidation degree can enhance the killing ability of CAR-T cells.

[0183] Figure 18 Part B shows the gene expression levels of CAR-T cells after different treatments. The hydrogel with 50% oxidation can induce a significant increase in the expression levels of CAR-T cell killing-related genes (IL2, IFNγ, TNFα), while ensuring that the expression levels of exhaustion-related genes (PD1, TIM3, CTLA4) do not increase significantly.

[0184] Therefore, 50% oxidation degree hydrogel is the optimal choice for culturing CAR-T cells. This hydrogel with preferred parameters can significantly enhance the cell-killing ability of CAR-T cells and has the potential to be applied to the treatment of pulmonary fibrosis.

[0185] Furthermore, through the examples, this application provides for the first time the unexpected activation effect of such hydrogels with specific preferred parameters (especially hydrogels with 50% oxidation degree and corresponding viscoelastic parameters) on cells with CAR proteins on the cell membrane.

[0186] In this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0187] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0188] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing chimeric antigen receptor macrophages and / or chimeric antigen receptor T cells with enhanced cell-killing ability, characterized in that, include: Chimeric antigen receptor macrophages (CAR-M) and / or chimeric antigen receptor T cells (CAR-T) are cultured on hydrogels to obtain CAR-M and / or CAR-T cells with enhanced cell-killing capabilities. The hydrogel described herein has the following characteristics: The hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel. The gelatin-oxidized sodium alginate crosslinked hydrogel has an oxidation degree of 50%; The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1953-1120 Pa, the loss modulus is 112.0-42.81 Pa, the loss coefficient is 0.06945-0.03730, and the stress relaxation coefficient is 2915-2019 s.

2. The method according to claim 1, characterized in that, The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1317±185.4 Pa, the loss modulus is 66.34±13.81 Pa, the loss coefficient is 0.05±0.0061, and the stress relaxation coefficient is 2589±368.

7. Preferably, the energy storage modulus is 1317 Pa, the loss modulus is 66.34 Pa, the loss coefficient is 0.05, and the stress relaxation coefficient is 2589 s.

3. The method according to claim 1, characterized in that, The CAR-M and / or CAR-T described therein have the following characteristics: It has reduced membrane tension; CAR proteins are uniformly distributed on the membrane surface; It has an increased number of CAR protein monomers and dimers. Optionally, the CAR-M and / or CAR-T is FAP5-CAR M and / or anti-CD19 CAR-T.

4. The method according to claim 1, characterized in that, The gelatin-sodium oxidized alginate crosslinked hydrogel was prepared by the following steps: Sodium alginate was oxidized using sodium periodate to obtain oxidized sodium alginate with an oxidation degree of 50%. The oxidized sodium alginate and gelatin are mixed to obtain a pre-crosslinked hydrogel; and The initially cross-linked hydrogel is immersed in a calcium ion solution for secondary cross-linking to obtain the gelatin-sodium oxidized alginate cross-linked hydrogel.

5. A hydrogel for preparing CAR-M and / or CAR-T cells with enhanced cell-killing ability, characterized in that, The hydrogel described herein has the following characteristics: The hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel. The gelatin-oxidized sodium alginate crosslinked hydrogel has an oxidation degree of 50%; The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1953-1120 Pa, the loss modulus is 112.0-42.81 Pa, the loss coefficient is 0.06945-0.03730, and the stress relaxation coefficient is 2915-2019 s.

6. The hydrogel according to claim 5, characterized in that, Prepared by the following steps: Sodium alginate was oxidized using sodium periodate to obtain oxidized sodium alginate with an oxidation degree of 50%. The oxidized sodium alginate and gelatin are mixed to obtain a pre-crosslinked hydrogel; and The initially cross-linked hydrogel is immersed in a calcium ion solution for secondary cross-linking to obtain the gelatin-sodium oxidized alginate cross-linked hydrogel.

7. The hydrogel according to claim 5, characterized in that, The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1317±185.4 Pa, the loss modulus is 66.34±13.81 Pa, the loss coefficient is 0.05±0.0061, and the stress relaxation coefficient is 2589±368.7 s. Preferably, the energy storage modulus is 1317 Pa, the loss modulus is 66.34 Pa, the loss coefficient is 0.05, and the stress relaxation coefficient is 2589 s.

8. The use of a hydrogel-cultured CAR-M and / or CAR-T in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The hydrogel described herein has the following characteristics: The hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel. The gelatin-oxidized sodium alginate crosslinked hydrogel has an oxidation degree of 50%; The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1953-1120 Pa, the loss modulus is 112.0-42.81 Pa, the loss coefficient is 0.06945-0.03730, and the stress relaxation coefficient is 2915-2019 s. The CAR-M and / or CAR-T cells cultured in hydrogels have the following characteristics: It has reduced membrane tension; CAR proteins are uniformly distributed on the membrane surface; It has an increased number of CAR protein monomers and dimers.

9. A kit for preparing a medicament for treating pulmonary fibrosis, characterized in that, include: Hydrogels, wherein the hydrogels are used to culture CAR-M and / or CAR-T cells and have the following characteristics: The hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel. The gelatin-oxidized sodium alginate crosslinked hydrogel has an oxidation degree of 50%; The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1953-1120 Pa, the loss modulus is 112.0-42.81 Pa, the loss coefficient is 0.06945-0.03730, and the stress relaxation coefficient is 2915-2019 s. CAR-M and / or CAR-T, wherein the CAR-M and / or CAR-T are used to treat pulmonary fibrosis after being cultured in the hydrogel.

10. The use of hydrogels in enhancing the cell-killing ability of CAR-M and / or CAR-T cells, characterized in that, The hydrogel described herein has the following characteristics: The hydrogel is a gelatin-sodium oxidized alginate crosslinked hydrogel. The gelatin-oxidized sodium alginate crosslinked hydrogel has an oxidation degree of 50%; The viscoelastic parameters of the gelatin-sodium alginate crosslinked hydrogel are as follows: The energy storage modulus is 1953-1120 Pa, the loss modulus is 112.0-42.81 Pa, the loss coefficient is 0.06945-0.03730, and the stress relaxation coefficient is 2915-2019 s. The CAR-M and / or CAR-T cells cultured in the hydrogel have the following characteristics: It has reduced membrane tension; CAR proteins are uniformly distributed on the membrane surface; It has an increased number of CAR protein monomers and dimers. Optionally, the CAR-M and / or CAR-T is FAP5-CAR M and / or anti-CD19 CAR-T.

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