Adapter hydrogel combined modular CAR-T product and application thereof in treatment of rheumatoid arthritis

Abstract

The invention discloses an adapter hydrogel combined modular CAR-T product and application thereof in treatment of rheumatoid arthritis, and relates to the technical field of biological medicine. According to the invention, a fluorescent molecule FITC is used for modifying antibodies / fusion proteins (especially CTLA4Ig, alphaCD19 and alphaCD4) aiming at different targets of immune cells, and an adapter library is constructed. The adapter and the temperature-sensitive copolymer are mixed and then subcutaneously injected, the mixed solution is gelatinized in situ, and the adapter is slowly and slowly released in vivo, so that the enrichment of the adapter at the inflammation part is improved. Meanwhile, CAR-T capable of recognizing FITC is prepared, and three adapters with the strongest curative effect, namely CTLA4-FITC, alphaCD19-FITC and alphaCD4-FITC, are screened out from a mouse model of collagen-induced arthritis (CIA). The adapter combination can further enhance the curative effect of RA, and provides a new platform for the treatment of autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an adapter hydrogel combined with modular CAR-T product and its application in the treatment of rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease (AID) characterized by synovial inflammation, immune cell infiltration, and joint destruction. Although traditional synthetic (e.g., methotrexate) and biologic (e.g., TNF-α inhibitor) antirheumatic drugs (DMARDs) have significantly improved the efficacy of RA, the overall response rate and long-term remission rate remain low.

[0003] Chimeric antigen receptor (CAR)-T cell therapy has made breakthrough progress in the treatment of autoimmune diseases such as systemic lupus erythematosus and RA. CAR-T cells targeting CD19 achieve drug-free remission in refractory systemic lupus erythematosus (SLE) by eliminating pathogenic B cells. Chimeric autoantibody receptor (CAAR)-T cells designed for anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis can specifically eliminate B cells that produce pathogenic antibodies. The pathogenesis of RA involves B cells, macrophages, CD4 + Abnormal activation of multiple immune cells such as T cells. However, the current CAR-T for autoimmune disease has a single target and cannot eliminate multiple autoimmune cells at the same time. Currently, no effective multi-target CAR-T product for the treatment of rheumatoid arthritis has been reported. Summary of the Invention

[0004] To address the above technical issues, the present invention provides an adaptor hydrogel combined with a modular CAR-T product for the efficient treatment of rheumatoid arthritis. The product of the present invention has the following advantages: 1) Flexible targeting, coordinated administration can eliminate a variety of immune cells. 2) The sustained-release thermosensitive gel significantly enhances the accumulation of the adaptor at the lesion site. 3) Because FITC is not expressed in the body, stopping the adaptor injection can terminate the killing effect of the CAR-T, thereby spatiotemporally regulating the efficacy of the CAR-T and achieving greater safety.

[0005] The first object of the present invention is to provide an adapter hydrogel combined with a modular CAR-T product, comprising:

[0006] A liquid preparation containing an adapter, wherein the adapter contains an antibody or fusion protein modified with a marker molecule, wherein the antibody or fusion protein specifically recognizes a target cell surface marker;

[0007] A liquid preparation containing a gel precursor solution containing a gel matrix for forming a temperature-sensitive gel, for forming a gel subcutaneously at the injection site;

[0008] A liquid preparation containing CAR-T cells, wherein the chimeric antigen receptor (CAR) in the CAR-T cells targets the marker molecule.

[0009] Furthermore, the gel matrix gels at 37° C. or body temperature; the gel matrix comprises a copolymer of polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid.

[0010] Furthermore, in the gel precursor solution, the concentration of the gel matrix is 15-30% (w / v).

[0011] Furthermore, in the adapter, the ratio of the number of the connected labeling molecules to the antibody or fusion protein is (0.5-5):1.

[0012] Furthermore, the labeling molecule includes a fluorescent molecule; the fluorescent molecule includes FITC.

[0013] Furthermore, the antibody or fusion protein is preferably one or more of αCD4 antibody, αCD19 antibody, and CTLA4Ig fusion protein.

[0014] Furthermore, the CAR-T cells are obtained by transfecting T cells with chimeric antigen receptors; the chimeric antigen receptors contain a coding sequence for a marker molecule antibody scFv.

[0015] Furthermore, the labeled molecule antibody scFv includes αFITC scFv; the coding sequence of the αFITC scFv is shown in SEQ ID NO.1.

[0016] Furthermore, the chimeric antigen receptor also contains a hinge region coding sequence, a transmembrane region coding sequence and an intracellular signal region coding sequence.

[0017] Furthermore, the hinge region includes a CD28 hinge region.

[0018] Furthermore, the transmembrane region includes a CD28 transmembrane region.

[0019] Furthermore, the intracellular signaling region contains a costimulatory molecule region and a signal transduction molecule region; the costimulatory molecule region includes a CD28 intracellular signaling region, and the signal transduction molecule region includes a CD3zeta intracellular signaling region.

[0020] Furthermore, the chimeric antigen receptor is connected to a tag sequence after the CD3zeta intracellular signal region, such as a Thy1.1 coding sequence.

[0021] Furthermore, the CD3zeta intracellular signaling region coding sequence and the Thy1.1 coding sequence are connected via a 2A peptide.

[0022] The second object of the present invention is to provide a pharmaceutical composition comprising:

[0023] At least one antibody or fusion protein modified with a marker molecule, wherein the antibody or fusion protein specifically recognizes a target cell surface marker; the at least one antibody or fusion protein modified with a marker molecule includes an αCD4 antibody modified with a marker molecule, an αCD19 antibody modified with a marker molecule, and a CTLA4 Ig fusion protein modified with a marker molecule;

[0024] A gel matrix, wherein the gel matrix is a thermosensitive gel matrix that forms a gel at body temperature (e.g., 35-40° C.);

[0025] An immune cell containing a chimeric antigen receptor that targets the marker molecule.

[0026] Furthermore, the gel matrix comprises a copolymer of polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid.

[0027] Furthermore, the immune cells include but are not limited to T cells, NK cells, etc.

[0028] The third object of the present invention is to provide the use of the adapter hydrogel combined with a modular CAR-T product or the pharmaceutical composition in the preparation of a pharmaceutical product.

[0029] Furthermore, the pharmaceutical products include products for treating autoimmune diseases, especially products for treating rheumatoid arthritis.

[0030] The fourth object of the present invention is to provide a drug for treating rheumatoid arthritis, comprising the adapter hydrogel combined with a modular CAR-T product or the pharmaceutical composition.

[0031] By means of the above solution, the present invention has at least the following advantages:

[0032] The present invention constructs adapters targeting different immune cells, combined with modular CAR-T, for multi-target synergistic treatment of RA. First, targeting the important immune cells in the pathogenesis of RA, the fluorescent molecule FITC functionalized with N-hydroxysuccinimide (FITC-PEG-NHS) is used to modify antibodies or fusion proteins targeting these cells to prepare adapters. The adapter is mixed with the thermosensitive copolymer polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid (PLGA-PEG-PLGA), which can quickly form a gel at 37°C ( Figure 1 A). Subcutaneous injection of the mixture can quickly gel at body temperature to form an adapter "reservoir", which slowly releases the adapter in the body and significantly increases its accumulation at the site of inflammation ( Figure 1B). On the other hand, a modular CAR-T targeting FITC (αFITC-CAR-T) was constructed and combined with an adapter hydrogel to eliminate pathogenic autoimmune cells in lesions such as inflamed joint cavities, effectively treating RA ( Figure 1 B). Finally, different adapters were screened and combined to obtain an adapter hydrogel combined with modular CAR-T product for the efficient treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0034] Figure 1 Schematic diagram of the hydrogel adapter combined with modular CAR-T for the efficient treatment of rheumatoid arthritis. (A) Synthesis route of the thermosensitive hydrogel adapter. (B) Mechanism of immune cell killing by the adapter combined with αFITC-CAR-T.

[0035] Figure 2 Preparation and characterization of modular CAR-T and adapters. (A) Schematic diagram of the αFITC-CAR gene sequence. (B) CD8 + Flow cytometric histogram of T cell positivity. (C) Schematic diagram of adapter synthesis. (D) Number of FITC connections for each adapter at different feed ratios. (E) Schematic diagram of the CTLA4-FITC bioactivity validation experiment. Produced by BioRender. (F) Experimental flow chart. (G) Representative flow cytometric plots of CTLA4-FITC binding to CD80 and CD86. (H) Mean fluorescence intensity of CD80 and CD86. Data are expressed as mean ± SD and analyzed using one-way ANOVA with Tukey's post hoc test. ns indicates no significant difference, **P < 0.01.

[0036] Figure 3 Figure 3 shows the in vitro cytotoxicity of different adapters combined with αFITC-CAR-T cells against target spleen cells. (A) Experimental flow chart. (B) Adapters CTLA4-FITC, αCD19-FITC, αCD4-FITC, αCD115-FITC, and αCD80-FITC kill CD11c + 、B220 + 、CD4 + T, CD11b + 、CD11c + Representative flow cytometry plots of cells. (C) Remaining number of each target cell. Data are expressed as mean ± SD and analyzed using one-way ANOVA with Tukey's post hoc test. ns indicates no significant difference; ****P < 0.0001.

[0037] Figure 4 Characterization and inflammation targeting of the hydrogel adapter. (A) Gelation of hydrogels with different mass-to-volume ratios (w / v%) at 37°C. (B) In vitro release kinetics of the hydrogel adapter. (C) Flowchart of the inflammation targeting experiment of CTLA4-Cy5 in the ZIA mouse model using different delivery methods. (D) Fluorescence images of mice at different time points. (E) Quantification of fluorescence intensity at the inflammatory site. (F) Area under the fluorescence intensity curve at the inflammatory site. Data are expressed as mean ± SD and analyzed using two-way ANOVA with Tukey's post hoc test (B) or one-way ANOVA with Tukey's post hoc test (F). ns indicates no significant difference; ****P < 0.0001.

[0038] Figure 5 Figure 1: Synergistic efficacy of the hydrogel-enhanced adapter and αFITC-CAR-T in CIA mice. (A) Experimental flow chart. (B) Arthritis score. (C) Body weight change. (D) Paw thickness. (E) Maximum rotation speed. (F) Running time. (G) Levels of various inflammatory factors in peripheral blood. (H) Flow chart of the spleen restimulation experiment. (I) Levels of the inflammatory factor IFN-γ after antigen restimulation of spleen cells. Data are expressed as mean ± SD and analyzed using two-way ANOVA with Tukey's post hoc test (B, C) or one-way ANOVA with Tukey's post hoc test (D, G, I). ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0039] Figure 6 The immune effect of hydrogel adapter combined with αFITC-CAR-T. (A) Synovial B cells, APCs and CAR-T cells in CD45 + Representative flow cytometry plots of cells. Numbers of B cells, APCs, and CAR-T cells in (B) synovium, (C) lymph nodes, and (D) spleen. Data are expressed as mean ± SD (n = 6) and analyzed using one-way ANOVA with Tukey's post hoc test. ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0040] Figure 7Comparison of the efficacy of different adapters combined with αFITC-CAR-T in CIA mice. (A) Experimental flow chart. (B) Arthritis score. (C) Body weight change. (D) Paw thickness. (E) Maximum rotational speed. (F) Running time. (G) Peripheral blood inflammatory cytokine levels of IL-6 and IL-1β. (H) Splenic cell inflammatory cytokine IFN-γ level after antigen restimulation. Data are expressed as mean ± standard deviation and analyzed using two-way ANOVA with Tukey's post hoc test (B, C) or one-way ANOVA with Tukey's post hoc test (D, H). ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0041] Figure 8 The immune effects of different adapters combined with αFITC-CAR-T on CIA mice. (A) Synovial B cells, CD4 + T cells, APCs and CAR-T cells in CD45 + Representative flow cytometry plots of cells. B cells, CD4 + The number of T cells, APCs, and CAR-T cells in (B) the synovium, (C) lymph nodes, and (D) the spleen. Data are expressed as mean ± standard deviation and analyzed using one-way ANOVA with Tukey's post hoc test. ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0042] Figure 9 The efficacy of the adapter combination combined with αFITC-CAR-T in CIA mice. (A) Experimental flow chart. (B) Arthritis score. (C) Body weight change. (D) Paw photograph. (E) Paw thickness. (F) Maximum rotational speed. (G) Running time. (H) Synovial inflammatory cytokines IL-6 and IL-1β levels. (I) Splenic cell inflammatory cytokine IFN-γ levels after antigen restimulation. Data are expressed as mean ± standard deviation and analyzed using two-way ANOVA with Tukey's post hoc test (B, C) or one-way ANOVA with Tukey's post hoc test (EI). ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0043] Figure 10Comparison of the therapeutic effects of each component of the combination therapy on CIA mice. (A) Experimental flow chart. (B) Arthritis score. (C) Body weight change. (D) Paw thickness. (E) Maximum rotational speed. (F) Running time. (G) Synovial proinflammatory cytokine levels. (H) Serum proinflammatory cytokine levels. (I) Levels of the inflammatory factor IFN-γ after restimulation of spleen cells with antigen. (J) Micro-CT images of mouse paws and coronal sections of ankle joints. Data are expressed as mean ± SD and analyzed using two-way ANOVA with Tukey's post hoc test (B, C) or one-way ANOVA with Tukey's post hoc test (D 1). ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0044] Figure 11 The immune effects of each component of the combined therapy on CIA mice. (A) Synovial B cells, CD4 + T cells, APCs and CAR-T cells in CD45 + Representative flow cytometry plots of cells. B cells, CD4 + The number of T cells, APCs, and CAR-T cells in (B) the synovium, (C) lymph nodes, and (D) the spleen. Data are expressed as mean ± standard deviation and analyzed using one-way ANOVA with Tukey's post hoc test. ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0045] Figure 12 To investigate the safety of the combined therapy, blood biochemistry and routine blood tests were performed after treatment with each component. Data are expressed as mean ± standard deviation. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0047] The materials and methods involved in the embodiments of the present invention are as follows:

[0048] (1) Reagents

[0049] PLGA-PEG-PLGA (LA:GA = 75:25) was purchased from Xi'an Ruixi Biotechnology Co., Ltd. Human CTLA4 Ig fusion protein (CTLA4, clone: CTLA-4-lg (hum. / hum.)), mouse CD19 monoclonal antibody (αCD19, clone: 1D3), mouse CD4 monoclonal antibody (αCD4, clone: GK1.5), mouse CD115 antibody (αCD115, clone: AFS98), mouse CD80 antibody (αCD80, clone: 16-10A1), mouse CD28 antibody (αCD28, clone: D665), and mouse CD3 antibody (αCD3, clone: 145-2C11 f(ab')2 fragments) were purchased from BioXCell, USA. Incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), and bovine type II collagen were purchased from Chondrex, USA. Zymosan A was purchased from Shanghai McLean Biochemical Technology Co., Ltd. Mouse IL-2, IL-7, and recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from Pepro Tech, USA.

[0050] (2) Experimental cells and animals

[0051] HEK-293T cells were purchased from Zhejiang Zhongchu Biotechnology Co., Ltd. BMDCs were extracted from the femoral and tibia bone marrow of C57BL / 6 mice. Male SPF-grade C57BL / 6 mice and DBA / 1 mice (both 6 to 8 weeks old) were purchased from Changzhou Cavens Laboratory Animal Company. The mice were kept in an environment with a temperature of 20±2°C, a humidity of 50±5%, and 12 hours of light per day. Before the experiment officially begins, the mice need to undergo an adaptive feeding phase of 1 to 3 days. All animal experimental projects are carried out under the strict review and supervision of the Animal Experiment Ethics Committee of Soochow University to ensure that every step of the experimental operation complies with the principles of experimental animal welfare and the ethical standards of Soochow University experimental animals.

[0052] The sequence information involved in the embodiments of the present invention is as follows:

[0053] The CAR sequence contains a series of αFITC scFv (extracellular antigen binding region), CD28 hinge (hinge region), CD28 TM (transmembrane region), CD28 costimulatory region, CD3zeta intracellular domain, P2A peptide, and Thy1.1. The amino acid sequences of each part are as follows:

[0054] αFITC scFv (SEQ ID NO. 1):

[0055] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDD AKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSS

[0056] CTLA4: purchased from BioXcell, Clone: CTLA-4-Ig (hum. / hum.), catalog number: BE0099.

[0057] αCD19: purchased from BioXcell, Clone: 1D3, Catalog No.: BE0150.

[0058] αCD4: purchased from BioXcell, Clone: GK1.5, catalog number: BE0003-1.

[0059] αCD115: purchased from BioXcell, Clone: AFS98, Catalog No.: BE0213.

[0060] αCD80: purchased from BioXcell, Clone: 16-10A1, Catalog No.: BE0024.

[0061] Example 1

[0062] (1) Preparation of modular CAR-T

[0063] The three-plasmid system was used to construct lentivirus. The coding sequences of αFITC scFv (clone FMC63), mouse CD28 hinge and transmembrane region, mouse CD28 co-stimulatory region, mouse CD3zeta intracellular domain, P2A and Thy1.1 were fused into the lentiviral vector to form the CAR plasmid. The CAR plasmid, packaging and envelope plasmids were amplified using Escherichia coli. Hieff HEK293T cells were transfected with Liposomal transfection reagent and the CAR, packaging, and envelope plasmids at a ratio of 3:2:1. Virus-containing supernatants were collected at 48 and 72 hours and centrifuged at 1000 × g for 5 minutes at 4°C. The supernatants were filtered through a 0.45 μm cellulose acetate membrane. The virus was concentrated by ultracentrifugation at 25,000 × g for 2 hours at 4°C, resuspended in serum-free medium, and stored at -80°C.

[0064] Extraction of CD8 from mouse spleen + T cells. Male C57BL / 6 or DBA / 1 mice were euthanized with carbon dioxide, and the spleens were dissected and passed through a 70 μm cell strainer. Cell pellets were collected by centrifugation at 700 g for 5 min, and red blood cell lysis buffer was added for incubation at room temperature for 3 min. Lysis was terminated by dilution with PBS. The cell suspension was filtered through a 40 μm cell strainer and centrifuged again (700 × g, 5 min). The supernatant was discarded and the cells were resuspended in PBS. EasySep TM Mouse CD8 + CD8 T cells were purified by T cell isolation kit + T cells. CD8 + T cells were resuspended in T cell-specific culture medium containing mouse IL-2 (20 ng / mL) and IL-7 (10 ng / mL), transferred to a culture dish pre-coated with αCD3 and αCD28, and placed in a 37°C incubator for activation for 3 days.

[0065] Lentiviral transfection of CD8 + T cell preparation αFITC-CAR-T. Activated CD8 + T cells were mixed with virus solution and the cell density was regulated to 5×10 6 Cells were plated at 400 μg / mL and Polybrene was added to a final concentration of 8 μg / mL. Twelve hours after infection, an equal volume of T cell-specific culture medium containing mouse IL-2 and IL-7 was added and cultured for a further 36 hours. The prepared αFITC-CAR-T cells were harvested by centrifugation and stained with αThy1.1-APC for transfection efficiency analysis by flow cytometry.

[0066] αFITC CAR structural sequence Figure 2 As shown in A. A three-plasmid system containing CAR, pMD2.G, and pSPAX2 was used to prepare lentivirus in HEK293T cells, which was then concentrated and transfected into T cells to prepare αFITC-CAR-T. The CAR transfection effect was detected by the tag protein Thy1.1. Flow cytometry results showed that the cell Thy1.1 positive rate reached 91.9% ( Figure 2 B).

[0067] (2) Preparation of adapter

[0068] FITC modified different types of biological preparations to prepare adapters. FITC-PEG 2000 -NHS was dissolved in anhydrous ethanol to prepare 100 mg / mL. 2000The FITC-PEG-NHS molar ratio was 1:2, 1:4, 1:6, 1:8, and 1:10 in a low protein adsorption tube, PBS was added to make the final protein concentration 1 mg / mL, and the pH was adjusted to 8.5. After reacting on a four-dimensional rotator for 4 hours at room temperature, the sample was added to a 50 kDa ultrafiltration tube and ultrafiltered 5 times with PBS to remove free FITC-PEG. 2000 -NHS. NanoDrop was used to detect the absorbance of the product at A280 to determine the protein concentration. A UV spectrometer was used to detect the absorbance of the FITC group at 495 nm. 2000 FITC concentration was determined using a standard curve constructed with -NHS. The number of FITC-linked molecules for each biological preparation was calculated according to Equation 1.

[0069]

[0070] Formula 1: Number of FITC-linked molecules per biological agent

[0071] (C FITC FITC-PEG 2000 -NHS concentration; C Biologics is the substance concentration of the biological agent)

[0072] FITC-PEG was synthesized by amide reaction. 2000 -NHS was coupled to the amino groups of the fusion protein CTLA4 Ig or monoclonal antibodies (αCD19, αCD4, αCD115 and αCD80) to prepare different adapters, namely CTLA4-FITC, αCD19-FITC, αCD4-FITC, αCD115-FITC and αCD80-FITC ( Figure 2 C). The absorbance of FITC group at 495nm was detected by UV spectrophotometer, and the number of FITC connected in each adapter was calculated. 2000 As the ratio of -NHS increases, the number of FITC molecules connected to the adapter gradually increases. In order to control the number of FITC molecules connected to different target adapters to be 2 (too many connections may affect the activity of antibodies or fusion proteins), CTLA4, αCD19, αCD4, αCD115, αCD80 and FITC-PEG 2000 -NHS feed molar ratios were 1:4, 1:4, 1:4, 1:6 and 1:6 to synthesize the adapter ( Figure 2 D).

[0073] Example 2

[0074] CTLA4-FITC biological activity verification:

[0075] In order to explore the FITC-PEG2000 The effect of modification on the biological activity and function of CTLA4 was verified by flow cytometry. The shielding effect of CTLA4-FITC on cellular CD80 / 86 was verified. BMDCs were stimulated with lipopolysaccharide (LPS) at a concentration of 100 ng / mL for 24 hours to induce their maturation. Fresh culture medium was then replaced, and CTLA4-FITC or unmodified CTLA4 (final protein concentration of 5 μg / mL) was added. After incubation for 4 hours, cells were collected and stained with flow cytometry antibodies αCD80-APC and αCD86-APC-Cy7. The fluorescence intensity of BMDCs was measured by flow cytometry.

[0076] CTLA4 can specifically bind to the co-stimulatory molecules CD80 / 86 on the surface of dendritic cells (DCs). To evaluate the effect of FITC modification on the biological activity of CTLA4, flow cytometry antibodies were used to compete with CTLA4-FITC for binding to CD80 / 86 on the surface of antigen-presenting cells to verify its biological activity ( Figure 2 E). BMDCs highly expressed CD80 / 86 after 24 hours of LPS stimulation. They were then divided into three groups: group G1 was treated with PBS, group G2 was treated with unmodified CTLA4, and group G3 was treated with CTLA4-FITC. After 4 hours of co-incubation, the cells were stained with flow cytometry antibodies αCD80-APC and αCD86-APC-Cy7, and the fluorescence intensity of cellular APC and APC-Cy7 was measured by flow cytometry ( Figure 2 F). The results showed that the fluorescence intensity of APC and APC-Cy7 in group G1 was significantly higher than that in groups G2 and G3, while there was no significant difference between groups G2 and G3, indicating that there was no statistical difference between CTLA4-FITC and CTLA4 in their ability to bind to CD80 / 86, proving that the modification of CTLA4 with two FITC molecules did not affect its biological activity ( Figure 2 G, H).

[0077] Example 3

[0078] In vitro killing of target cells by adapter combined with αFITC-CAR-T:

[0079] Mouse spleen cells were seeded into 48-well plates and incubated with different adaptors (CTLA4-FITC, αCD19-FITC, αCD4-FITC, αCD115-FITC, and αCD80-FITC) for 2.5 hours. αFITC-CAR-T cells were then added at a 10:1 ratio of spleen cells to CAR-T cells and cultured for 24 hours. All cells were then harvested, and counting beads were added to each well. Target cell numbers were determined by flow cytometry using antibodies against αCD11c-APC, αB220-PE, αCD4-APC, αCD11b-APC, and αThy1.1-PE-Cy7.

[0080] αFITC-CAR-T and different adapters (CTLA4-FITC, αCD19-FITC, αCD4-FITC, αCD115-FITC, αCD80-FITC) were added to the mouse spleen cell solution. Group G1 cultured spleen cells alone, group G2 co-cultured spleen cells with αFITC-CAR-T, and group G3 co-cultured spleen cells with different adapters and then added with αFITC-CAR-T. Figure 3 A). The results showed that the number of target cells in the G3 group was the lowest among all the adapters. Compared with the G1 group, the number of target cells in the G3 group with the addition of CTLA4-FITC, αCD19-FITC, αCD4-FITC, αCD115-FITC, and αCD80-FITC decreased by 78.1%, 56.7%, 78.3%, 74.1%, and 80.6%, respectively. There was no significant difference in the number of target cells between the G1 and G2 groups ( Figure 3 B, C). The above results show that αFITC-CAR-T can achieve precise killing of target cells by combining with different adapters; while αFITC-CAR-T alone cannot kill target cells because it cannot recognize target cells.

[0081] Example 4

[0082] (1) Preparation of PLGA-PEG-PLGA hydrogel

[0083] At room temperature of 25°C, PLGA-PEG-PLGA (PPP) polymer was accurately weighed and placed in a glass bottle. The corresponding volume of PBS was added to prepare solutions with mass concentrations of 16, 20, 24, and 28 w / v%. The solutions were placed on a four-dimensional rotator and rotated until the polymer was completely dissolved.

[0084] At 25°C, invert the glass bottles containing the PPP solutions of varying concentrations and photograph them. Then, place them in a metal bath and gradually heat the temperature from 25°C to 37°C. After stabilizing at 37°C for 10 minutes, invert the bottles and observe for 30 seconds. If no liquid flows downward, gelation has occurred. Once gelation has occurred, invert the bottles and photograph them.

[0085] At room temperature (25°C), PPP polymer solutions of 16, 20, 24, and 28 w / v% all appeared liquid. When the temperature gradually increased to 37°C, PPP solutions of all concentrations transformed into a colloidal state and lost their fluidity ( Figure 4 A).

[0086] (2) In vitro release kinetics of adapter hydrogels

[0087] CTLA4-FITC was diluted with PBS to a protein concentration of 0.2 mg / mL. Fluorescence intensity was measured using a microplate reader as total fluorescence intensity (excitation wavelength 480 nm, emission wavelength 530 nm). PPP polymer was weighed at room temperature (25°C) and dissolved in PBS containing 0.2 mg / mL CTLA4-FITC to prepare CTLA4-FITC@Gel with 16 and 28 w / v PPP. After complete dissolution, the gel was formed at 37°C and then transferred to a 200 kDa dialysis bag. The dialysis bag containing CTLA4-FITC@Gel was placed in a 50 mL centrifuge tube filled with PBS and subjected to constant temperature shaking at 37°C and 100 rpm for 7 days. At predetermined time points, 200 μL of release medium was aspirated from the centrifuge tube and replaced with 200 μL of PBS at the same temperature. The fluorescence intensity of the samples was measured using a microplate reader. The cumulative release percentage of CTLA4-FITC was calculated according to Equation 2, and the in vitro release curve was plotted.

[0088]

[0089] Formula 2: Calculation formula for the cumulative release percentage of CTLA4-FITC

[0090] (E r is the cumulative release percentage of CTLA4-FITC; V e is the replacement volume of the release medium, i.e. 200 μL; V0 is the total volume of the release medium, i.e. 50 mL; F CTLA4-FITC is the total fluorescence of CTLA4-FITC in the initial dialysis bag; f i is the amount of fluorescence released during the i-th replacement sampling)

[0091] Taking CTLA4-FITC as an example, the in vitro release kinetics of the adapter hydrogel were investigated. After loading CTLA4-FITC into 16 w / v% or 28 w / v% hydrogel, it was placed in a dialysis bag with a molecular weight cutoff of 200 kDa and the release kinetics were investigated in normal saline at 37°C. CTLA4-FITC has a molecular weight of approximately 105 kDa and can pass through the dialysis bag. After one day, 87.5% of the CTLA4-FITC in the free group diffused out of the dialysis bag ( Figure 4B). When CTLA4-FITC is loaded into the hydrogel, its release rate is affected by the density of the hydrogel network structure, and the cross-linking density of the hydrogel is positively correlated with the concentration of the thermosensitive polymer in the mixed solution. High concentrations of polymers will cause the pore size of the formed hydrogel to decrease and the network structure to become denser, thereby limiting the diffusion of the loaded drug molecules and slowing down their release rate. In the present invention, the cumulative release rate of each group was basically the same on the 10th day, indicating that the total amount of CTLA4-FITC released by the two hydrogel groups was equivalent during the long-term release process. However, when the polymer increased from 16w / v% to 28w / v%, the release rate of CTLA4-FITC decreased. The time required for the 16w / v% and 28w / v% drug-loaded hydrogel groups to release 50% of the drug load was 1 day and 3.5 days, respectively ( Figure 4 B). Therefore, the 28w / v% PPP thermosensitive hydrogel has a stronger sustained-release effect than the 16w / v% PPP thermosensitive hydrogel.

[0092] Example 5

[0093] Inflammatory targeting of CTLA4-FITC by different delivery methods:

[0094] The inflammatory targeting properties of CTLA4-FITC under different delivery methods were verified in the ZIA mouse model using a small animal imaging system. Given that FITC fluorescence is susceptible to background interference, CTLA4-Cy5 was prepared for in vivo distribution studies. First, CTLA4-Cy5@Gel with PPP concentrations of 16 and 28 w / v% were prepared, in which the CTLA4 concentration was 0.4 mg / mL. Zymosan A was dissolved in PBS to prepare a 15 mg / mL suspension. The suspension was sonicated with a probe for 30 minutes, then boiled for 15 minutes and placed at room temperature until use. Male C57BL / 6 mice were anesthetized with the inhalation anesthetic isoflurane, fixed in a supine position, and the right knee joint area was depilated. 20 μL of Zymosan A solution was injected into the knee joint cavity to establish the ZIA mouse model (zymosan A-induced arthritis model). Twenty-four hours after modeling, mice were randomly divided into four groups: G1: CTLA4-Cy5 (iv); G2: CTLA4-Cy5 (sc); G3: CTLA4-Cy5@Gel (16 w / v%) (sc); and G4: CTLA4-Cy5@Gel (28 w / v%) (sc). Each mouse was injected with 40 μg of CTLA4 protein in a 100 μL solution. Intravenous injections were performed via the tail vein, and subcutaneous injections were performed on the skin on both sides of the back. Fluorescence intensity was monitored using an IVIS imager (excitation wavelength: 620 nm, emission wavelength: 680 nm) at 3, 6, 9, 12, 24, 48, 72, 120, and 144 hours after injection.

[0095] To explore the effects of different administration methods on the inflammatory targeting of the adapter, a ZIA model was established, with the following groups: G1 group received intravenous injection of CTLA4-Cy5; G2 group received subcutaneous injection of CTLA4-Cy5; G3 group received subcutaneous injection of CTLA4-Cy5@Gel (16w / v%); G4 group received subcutaneous injection of CTLA4-Cy5@Gel (28w / v%) ( Figure 4 C). The results showed that the fluorescence intensity of the right knee joint (inflammation site) in group G1 was the highest at 3 hours, and in group G2 it peaked at 12 hours, and then the fluorescence intensity of both groups decreased rapidly; the trends of groups G3 and G4 were similar, with the fluorescence intensity peaking at 24 hours and then slowly decreasing, but group G4 still maintained a strong fluorescence intensity at 144 hours; the time for the fluorescence intensity to decrease to 50% after reaching the peak in the four groups was 12, 24, 72, and 120 hours, respectively ( Figure 4 D, 4E). The area under the curve (AUC) was calculated to evaluate the enrichment of CTLA4-Cy5 in inflamed joints. The AUC of group G2 was slightly higher than that of group G1, but there was no statistical difference. The AUCs of groups G3 and G4 were significantly higher than those of group G2, indicating that loading the adapter into the hydrogel can enhance the inflammatory enrichment of the adapter. The AUC of group G4 was the highest, which was 2.2 times that of group G2 and 1.3 times that of group G3, indicating that the higher the concentration of the polymer forming the hydrogel, the stronger the inflammatory enrichment effect of the loaded adapter. The AUC of the inflamed joints in group G4 was 6.3 times that of the normal joints, proving that CTLA4-Cy5 was effectively enriched in the inflammatory site ( Figure 4 F) Therefore, loading CTLA4-Cy5 into hydrogels and then subcutaneously injecting them helps enhance the inflammatory accumulation of the adaptor.

[0096] Example 6

[0097] Effect of hydrogel delivery system on the therapeutic efficacy of combination therapy in CIA mouse model:

[0098] A CIA mouse model (collagen-induced arthritis model) was established. On day 0, bovine type II collagen and an equal volume of CFA were thoroughly mixed using a handheld high-speed homogenizer in an ice bath to prepare a water-in-oil emulsion. DBA / 1 mice were given a primary immunization with 100 μL of this emulsion injected intradermally at the base of their tails. On day 21, an emulsion of bovine type II collagen and IFA was prepared under the same conditions, and DBA / 1 mice were given a second booster immunization with 100 μL of this emulsion injected intradermally at the base of their tails.

[0099] When the paws of mice showed slight redness and swelling, they were divided into groups: G1: αCD19-FITC; G2: αCD19-FITC@Gel (16w / v%); G3: αFITC-CAR-T+αCD19-FITC; G4: αFITC-CAR-T+αCD19-FITC@Gel (16w / v%); G5: αFITC-CAR-T+CTLA4-FITC; G6: αFITC-CAR-T+CTLA4-FITC@Gel (16w / v%). Mice were treated on days 26, 30, 34, and 38. The subcutaneous injection dose of the adapter was 40 μg per mouse, and the intravenous injection dose of αFITC-CAR-T was 5×10 6 cells per mouse. αFITC-CAR-T cells were injected 12 hours after adapter injection. Starting on day 26, mice were observed daily, and body weight and RA scores were recorded. On day 43, paw thickness was measured using a vernier caliper in each group.

[0100] On day 43, mice underwent a rotating cage test to assess their locomotion and paw grip. Prior to evaluation, mice underwent acclimation training. After completing acclimation training, the mice were placed in a rotating cage and the speed was gradually increased to 70 rpm. The speed was recorded until the mice fell from the cage. Subsequently, the speed was fixed at 25 rpm, and the time the mice could maintain continuous movement within the cage was observed and recorded.

[0101] On day 43, after mice were anesthetized with isoflurane, blood samples were collected from the retroorbital venous plexus and placed in heparin-free centrifuge tubes. Subsequently, the mice were euthanized using the carbon dioxide method. The tubes containing the blood samples were allowed to stand at room temperature for 2 hours to ensure complete coagulation of the blood. The coagulated blood was centrifuged at 1200 × g for 20 minutes at 4°C. The supernatant serum was carefully separated and the concentrations of the proinflammatory cytokines IL-6, IL-1β, and TNF-α were measured according to the instructions of the ELISA kits.

[0102] After restimulation of spleen cells with antigens, the level of proinflammatory cytokine IFN-γ was measured. Splenocytes were resuspended in T cell culture medium containing mouse IL-2 (20 ng / mL) and IL-7 (10 ng / mL) to a concentration of 5 × 10 6 cells / mL, 0.5 mL was inoculated into each well of a 48-well plate, and antigen peptide collagen was added. 259-272 After 48 h of stimulation, the supernatant was collected and the IFN-γ concentration was determined using an ELISA kit.

[0103] The ankle synovium of mice was dissected and digested with 1% type IV collagenase and 0.2% DNase at 100 rpm and 37°C for 2 hours until the tissue was completely dissociated. The digested tissue fluid was passed through a 70 μm cell strainer, centrifuged at 300 × g for 5 minutes, and the supernatant was removed. The synovial single cell solution was resuspended in PBS. The number of various immune cells was determined by flow cytometry. Mouse lymphocytes and spleen cells were stained using the same method to determine the number of various immune cells.

[0104] Here are the results:

[0105] This study compared the efficacy of two adapters, CTLA4-FITC and αCD19-FITC, in CIA mice after being free and loaded into 16% w / v hydrogels. Figure 5 As shown in A.

[0106] (1) Efficacy evaluation:

[0107] On day 43, the RA index scores of mice in the αCD19-FITC@Gel group (G2) were not statistically different from those in the αCD19-FITC group (G1), demonstrating that αCD19-FITC loaded into hydrogels could not enhance the therapeutic efficacy of αCD19-FITC. The RA index of the αCD19-FITC combined with αFITC-CAR-T group (G3) was significantly decreased compared to the G1 group, demonstrating that the adapter combined with modular CAR-T can significantly reduce paw swelling ( Figure 5 B). The score of the αCD19-FITC@Gel combined with αFITC-CAR-T group (G4) was reduced by 54.8% compared with the αCD19-FITC combined with αFITC-CAR-T group (G3). Similarly, the RA score of the CTLA4-FITC@Gel combined with αFITC-CAR-T group (G6) was reduced by 38.3% compared with the free αCD19-FITC combined with αFITC-CAR-T group (G5). These results indicate that the hydrogel loaded with adapters can enhance the autoimmune cell killing efficiency of modular CAR-T ( Figure 5 B). The degree of weight loss in CIA mice was positively correlated with disease severity; the more severe the paw swelling, the more significant the weight loss. The weight curve results showed that the weight loss in the G6 group was significantly lower than that in the G5 group, and the weight loss in the G4 group was also lower than that in the G3 group, further demonstrating that hydrogel-loaded adapters can enhance the efficacy of αFITC-CAR-T ( Figure 5 C).

[0108] On the 43rd day, the paw thickness of the G4 group was the lowest, at 6.8 mm. The efficacy of the G4 group in reducing paw thickening was 2.0 times that of the G3 group, and the efficacy of the G6 group was 3.5 times that of the G5 group ( Figure 5D). The locomotion and paw gripping abilities of RA mice were negatively correlated with the severity of the disease. When the rotational cage speed gradually increased, the G4 and G6 groups showed the strongest effect; the maximum speed that the G4 group mice could maintain was 1.3 times that of the G3 group, and the G6 group was 1.2 times that of the G5 group ( Figure 5 E). When the fixed speed was 25 rpm, the mice in groups G4 and G6 still persisted the longest, which was 1.3 and 1.2 times that of the free adapter G3 and G5 groups ( Figure 5 F).

[0109] The serum pro-inflammatory cytokine levels showed that the G4 group had a better inhibitory effect on IL-6, TNF-α and IL-1β levels than the G3 group, and the G6 group had a stronger effect than the G5 group. Among them, the G4 group showed the strongest inhibitory effect on inflammatory factors ( Figure 5 G) Spleen cells using antigen peptide collagen 259-272 After 48 h of stimulation, the IFN-γ concentration in the supernatant was detected by ELISA ( Figure 5 H). Except for the G1 and G2 groups, which showed no significant difference in IFN-γ secretion levels, the IFN-γ secretion levels of the other adapters were all lower than that of the free adapter group; among them, the G4 group significantly inhibited IFN-γ secretion ( Figure 5 I).

[0110] Therefore, the hydrogel-loaded adapter combined with αFITC-CAR-T can significantly reduce paw swelling, inhibit disease progression, and restore mice's motor ability. The CTLA4-FITC@Gel combined with αFITC-CAR-T showed the best efficacy.

[0111] (2) Analysis of immune cells in synovium, lymph nodes, and spleen:

[0112] The results of mouse synovial immune cell analysis showed that the αCD19-FITC combined with αFIT-CAR-T group (G3) could reduce the number of B cells. The effect was strongest after the adapter was loaded into the hydrogel (G4), with the number reduced by 17.6% compared with the G3 group ( Figure 6 A, B). In addition, CTLA4-containing adapters (G5 and G6) can significantly reduce CD11b in synovium. + / CD11c + The APC number in the hydrogel G6 group was the best, with the number reduced by 15.4% compared with the G5 group ( Figure 6 A, B) αFITC-CAR-T in G4 and G6 groups showed an increase in CD45 + The proportion of cells in G3 and G5 groups was 1.5 and 1.4 times that of G3 and G5 groups, respectively, and the number was 1.5 and 1.9 times that of G5 groups, respectively. This proves that the hydrogel adapter library can significantly promote the proliferation of CAR-T and increase its number in the synovium compared with the free adapter. Figure 6 A, B).

[0113] The number of immune cells in lymph nodes was consistent with that in synovium. + B cells account for CD45 + The proportion of cells was the smallest, and the number was reduced by 30.0% compared with the G3 group. The clearance effect was significantly better than that of the G3 group ( Figure 6 C). At the same time, CTLA4 adaptors (G5 and G6) can reduce CD11b in lymph nodes. + / CD11c + The number of APCs, especially the G6 group, had the best clearance effect. Compared with the G5 group, the proportion of APCs decreased by 24.1% and the number decreased by 16.2% ( Figure 6 C). Analysis of αFITC-CAR-T in lymph nodes revealed that the proportion of αFITC-CAR-T in G4 and G6 groups was 1.9 and 2.1 times that of G3 and G5 groups, respectively, and the number was 1.4 and 1.7 times that of G3 and G5 groups, respectively, which once again proved that the hydrogel adapter can enhance CAR-T proliferation ( Figure 6 C).

[0114] The results of the analysis of various immune cells in the spleen were consistent with the trends of the above two tissues. + B cells account for CD45 + The number of cells was reduced by 33.2% compared with the G3 group, so the clearance effect of the G4 group was significantly better than that of the G5 group ( Figure 6 D). At the same time, CTLA4-containing adapters (G5 and G6) can reduce CD11b + / CD11c + In terms of the number of APCs, the G6 group had the best clearance effect. Compared with G5, the proportion of APCs decreased by 23.7% and the number decreased by 26.7% ( Figure 6 D). The proportion of αFITC-CAR-T in G4 and G6 groups was 2.4 and 3.4 times that of G3 and G5 groups, respectively, and the number was 1.8 and 2.5 times that of G3 and G5 groups, respectively. Figure 6 D).

[0115] In summary, compared with free adapters, the hydrogel adapter reservoir can significantly eliminate target cells, improve the in vivo proliferation of αFITC-CAR-T, and enhance the efficacy of RA.

[0116] Example 7

[0117] In vivo screening of different adapters:

[0118] A CIA mouse model (collagen-induced arthritis model) was established. After mild redness and swelling of the paws, the mice were grouped as follows: G1: Healthy; G2: RA; G3: αCD19-FITC@Gel; G4: CTLA4-FITC@Gel; G5: αCD4-FITC@Gel; G6: αCD80-FITC@Gel; G7: αCD115-FITC@Gel. Examples 6 and 7 were conducted on the same batch of animals. The data for G3 and G4 in this example are consistent with those for G4 and G6 in Example 6, respectively.

[0119] Mice were treated on days 26, 30, 34, and 38. The adapter was injected subcutaneously at a dose of 40 μg per mouse, and αFITC-CAR-T was injected intravenously at a dose of 5 × 10 6 cells per mouse. αFITC-CAR-T was injected 12 hours after adapter injection. The therapeutic efficacy was evaluated as described in Example 6.

[0120] Here are the results:

[0121] This example further evaluates the in vivo efficacy of modular CAR-T combined with different adapters to screen for more effective adapters. The experimental process is shown in Figure 7 A. Efficacy evaluation:

[0122] The RA index of the αCD19-FITC@Gel group (G3) was the lowest, which was 76.8% lower than that of the RA model group (G2). Figure 7 B). Compared with the model group, the weight loss of all mice in the adapter group was significantly improved, indicating that they all had a certain degree of therapeutic effect ( Figure 7 C). Based on RA scores and weight changes, the efficacy ranking from high to low was G3 > G5 ≈ G4 > G6 > G7, i.e., αCD19-FITC@Gel > αCD4-FITC@Gel ≈ CTLA4-FITC@Gel > αCD80-FITC@Gel > αCD115-FITC@Gel.

[0123] On the 43rd day, the paw thickness of the mice in the healthy group was 5.7 mm, and that of the model group was 17.7 mm. All the adapter groups were significantly lower than the model group. Among them, the paw thickness of the mice in the G3 group was the lowest, only 6.8 mm, which inhibited 61.5% of the paw thickening compared with the model group. The therapeutic effects were 4.6, 1.7, 1.2 and 1.1 times that of the αCD115-FITC@Gel group (G7), αCD80-FITC@Gel group (G6), αCD4-FITC@Gel (G5) and CTLA4-FITC@Gel (G4) groups, respectively. Figure 7D). The maximum rotation speed that mice in all treatment groups could tolerate in the rotating cage increased, with the G3 group performing the best, with the maximum tolerable rotation speed increased to 54.8 rpm, 4.4 times that of the model group ( Figure 7 E). Under the condition of a fixed speed of 25 rpm, the mice in group G3 performed the best among all treatment groups, with an exercise time of 59.5 s, which was 3.9 times that of the model group ( Figure 7 F) The ranking of efficacy based on paw thickness measurement and motor ability assessment was consistent with the above results.

[0124] The levels of inflammatory factors IL-6 and IL-1β in the serum of mice in the model group were the highest, and were significantly reduced in all treatment groups. Among them, the inhibitory effect of the G3 group was the strongest, inhibiting IL-6 secretion by 64.8% and IL-1β secretion by 49.8%, respectively. Figure 7 G). The order of inflammation inhibition effect of each group is consistent with the above results. All treatment groups can inhibit the secretion of IFN-γ by spleen cells after stimulation, among which the inhibitory effect of G3 group is the most significant ( Figure 7 H). The order of IFN-γ inhibitory efficacy is G3 > G4 > G5 > G6 > G7.

[0125] Synovial, lymph node, and spleen immune cell analysis:

[0126] B220 in the synovium of mice in the model group + B cells accounted for 36.3%. Among the treatment groups, the G3 group had the best effect on the clearance of B cells, with the number of B cells reduced by 38.5% compared with the model group ( Figure 8 A, B) G5 group CD4 + The T cell clearance effect was the best, with the proportion and number of T cells reduced by 37.8% and 37.2% respectively compared with the model group. Figure 8 A, B). Similarly, the G4 group in each treatment group showed a significant difference in CD11b + / CD11c + The APC clearance effect was the best, with the proportion reduced by 22.5% and the number reduced by 41.9% compared with the model group. Figure 8 A, B). Different adapter combinations have different activation effects on αFITC-CAR-T in the synovium. G4 group has the best effect in promoting CAR-T proliferation, followed by G5, G3, G6 and G7, namely CTLA4-FITC@Gel, αCD4-FITC@Gel, αCD19-FITC@Gel, αCD80-FITC@Gel and αCD115-FITC@Gel ( Figure 8 A, B).

[0127] Further analysis of immune cells in lymph nodes showed that the results were consistent with the trends in synovial tissue. Among the treatment groups, G3 group had the best effect on the clearance of B cells, with the number of B cells reduced by 54.8% compared with the model group ( Figure 8C) G5 group on CD4 + The clearance effect of T cells was the best, and the number was reduced by 44.3% compared with the model group ( Figure 8 C). In addition, the G4 group showed a significant difference in CD11b expression among the treatment groups. + / CD11c + The APC clearance effect was the best, and the number was reduced by 34.8% compared with the model group ( Figure 8 C). In the analysis of αFITC-CAR-T activation in lymph nodes, G4 group had the best effect in promoting αFITC-CAR-T proliferation, followed by G5, G3, G6 and G7 ( Figure 8 C).

[0128] Similarly, the results of spleen immune cell analysis were consistent with the above conclusions. Among the treatment groups, the G3 group had the best B cell clearance effect, with a 48.5% reduction in proportion and a 44.1% reduction in number compared with the model group ( Figure 8 D) At the same time, the G5 group in each treatment group had a negative effect on CD4 + The T cell clearance effect was the best, with the number of T cells reduced by 45.9% compared with the model group ( Figure 8 D) In addition, the G4 group showed a significant difference in CD11b expression among the treatment groups. + / CD11c + The APC clearance effect was the best, with the number of APCs decreasing by 39.7% compared with the model group ( Figure 8 D). Analysis of αFITC-CAR-T activation in the spleen showed that the G3 and G4 groups were significantly better than the other adapter groups ( Figure 8 D) The above results show that the adapters in groups G3, G5, and G4 have stronger efficacy, while those in groups G6 and G7 are relatively weaker.

[0129] In summary, a comprehensive evaluation was conducted based on RA scores, paw thickness, motor ability, inflammatory factor levels, and immune cell clearance, and finally αCD19-FITC, CTLA4-FITC, and αCD4-FITC adapters were screened for subsequent studies.

[0130] Example 8

[0131] Comparison of the therapeutic effects of various adapter hydrogels (with a fixed total amount of adapters) combined with αFITC-CAR-T on CIA mice

[0132] After slight redness and swelling appeared in the paws of CIA model mice, they were divided into groups: G1: Healthy; G2: RA; G3: αFITC-CAR-T+αCD4-FITC@Gel; G4: αFITC-CAR-T+αCD19-FITC@Gel; G5: αFITC-CAR-T+CTLA4-FITC@Gel; G6: αFITC-CAR-T+αCD19 / αCD4-FITC@Gel; G7: αFITC-CAR-T+αCD19 / CTLA4-FITC@Gel; G8: αFITC-CAR-T+αCD4 / CTLA4-FITC@Gel; G9: αFITC-CAR-T+αCD4 / CTLA4 / αCD19-FITC@Gel.

[0133] On days 28, 35, and 42, mice were treated with either 40 μg of adapter or 16 w / v% hydrogel containing 40 μg of adapter administered subcutaneously. If two different adapters were used, 20 μg of each was administered; if three different adapters were used, 13.4 μg of each was administered. Twelve hours after adapter injection, 5×10 6 αFITC-CAR-T. The experiment was ended on day 43 to evaluate the therapeutic effect.

[0134] (1) Three aptamer combinations:

[0135] Under the condition of a total dose of 40 μg, the selected three adapters, CTLA4-FITC, αCD19-FITC and αCD4-FITC, were combined and loaded into the hydrogel. The dose of the two adapters was 20 μg each, and the synergistic dose of the three adapters was 13.4 μg each. The experimental process is as follows: Figure 9 As shown in A.

[0136] The RA score of the dual-adapter αCD19 / αCD4 group (G6) was significantly lower than that of the single-adapter αCD4 group (G3) and the αCD19 group (G4); similarly, the RA score of the αCD19 / CTLA4 group (G7) was significantly lower than that of the αCD19 group (G4) and the CTLA4 group (G5); and the RA score of the αCD4 / CTLA4 group (G8) was significantly lower than that of the αCD4 group (G3) and the CTLA4 group (G5) ( Figure 9 B) The above results demonstrate that dual adapters have a better efficacy than a single adapter when the total dose is the same.

[0137] The RA score of the triple adapter synergistic αCD4 / CTLA4 / αCD19 group (G9) was significantly better than that of the three dual adapter synergistic groups (G6, G7 and G8). Figure 9B). The results of weight changes showed that mice in the G9 group had the lowest weight loss, further confirming that the triple adapter is more effective in inhibiting disease progression than using either adapter alone or in combination ( Figure 9 C).

[0138] The right hind paws of mice in the model group had severe lesions, with swelling in all joints including the ankle joint. The right hind paws of mice in the healthy group were normal in morphology and without swelling. The right hind paws of mice in the other treatment groups all had varying degrees of swelling, with only two toes of the right hind paws of mice in the G9 group having the mildest lesions ( Figure 9 D). The paw thickness of each treatment group was significantly lower than that of the model group. The paw thickness of the G9 group was the lowest, which was 60.1% less than that of the model group. The paw thickness of the G6, G7 and G8 groups were 40.8%, 48.9% and 34.4% less than that of the model group, respectively. Figure 9 E).

[0139] Rotating cage behavioral tests showed that the maximum rotation speed of the G9 group increased significantly to 61.5 rpm, which was 3.5 times that of the model group; the G6, G7 and G8 groups were 47.2, 53 and 45.5 rpm, respectively, which were 2.7, 3.0 and 2.6 times that of the model group ( Figure 9 F). When the speed was fixed at 25 rpm, the longest persistence time of the G9 group was 64.2 s, which was 3.3 times that of the model group; the G6, G7 and G8 groups were 2.6, 2.8 and 2.5 times that of the model group respectively. Figure 9 G) The results showed that at the same dose, the triple adapter could significantly improve the motor function of RA mice compared with either a single or dual adapter.

[0140] The G9 group inhibited 80.4% of the inflammatory factor IL-6 secretion, while the G6, G7, and G8 groups only inhibited 51.8%, 59.6%, and 42.1%, respectively. This indicates that the therapeutic effect of the triple adapter is significantly better than that of a single or dual adapter. The trend of the IL-1β determination results is consistent with that of IL-6 ( Figure 9 H). In addition, each treatment group could inhibit the secretion of IFN-γ after spleen cell antigen stimulation, among which the G9 group had the strongest inhibitory effect, with the IFN-γ level reduced by 72.9% compared with the model group, while the G6, G7 and G8 groups decreased by 50.4%, 59.5% and 46.0% respectively ( Figure 9 I), further indicating that the triple adapter can more effectively reduce the inflammatory response.

[0141] In summary, the synergistic use of CTLA4-FITC, αCD19-FITC, and αCD4-FITC adapters at the same dose can effectively treat RA, and the efficacy is better than that of a single or dual adapter.

[0142] Example 9

[0143] Comparison of the efficacy of adapter combination gel combined with αFITC-CAR-T and each component:

[0144] After slight redness and swelling appeared on the paws of CIA mice, they were divided into groups: G1: Healthy; G2: RA; G3: αCD4 / CTLA4 / αCD19; G4: αCD4 / CTLA4 / αCD19@Gel; G5: αFITC-CAR-T;

[0145] G6: αFITC-CAR-T+αCD4 / CTLA4 / αCD19-FITC; G7: αFITC-CAR-T+αCD4 / CTLA4 / αCD19-FITC@Gel, 6 mice in each group.

[0146] Mice were treated on days 26, 33, and 40. A total of 40 μg of αCD4 / CTLA4 / αCD19 adapters were injected, with 13.4 μg of each adapter. The hydrogel concentration was 28 w / v%, and the number of αFITC-CAR-T cells injected was 5 × 10 6 The experiment was ended on day 41 and the therapeutic effect was evaluated.

[0147] Here are the results:

[0148] (1) Efficacy evaluation

[0149] The experimental process is as follows Figure 10 A. The triple adapter (G3 and G4) can reduce the RA index in mice, and the effect is stronger after loading into the hydrogel ( Figure 10 B). The RA index of the αFITC-CAR-T group (G5) injected alone was almost the same as that of the model group (G2), proving that modular CAR-T cannot function without the participation of the adapter. The RA index of the αFITC-CAR-T+αCD4 / CTLA4 / αCD19-FITC group (G6) was significantly lower than that of the G3 group, proving that the adapter combined with CAR-T can effectively inhibit paw swelling. The efficacy of the αFITC-CAR-T+αCD4 / CTLA4 / αCD19-FITC@Gel group (G7) was further improved compared with the G6 group, and the RA index was reduced by 87.5% compared with the model group, proving that the hydrogel delivery system can enhance the synergistic efficacy of the adapter and αFITC-CAR-T ( Figure 10 B). The weight change curve also showed that the weight loss of mice in group G7 was the lowest ( Figure 10 C).

[0150] On day 41, the paw thickness of the mice in the healthy group was 6.6 mm. Among all the treatment groups, the paw thickness of the mice in the G7 group was the smallest, at 7.0 mm, which was almost the same as that of the healthy group. The paw thickness was 40.2% of that of the model group ( Figure 10D). All triple adapter groups (G3, G4, G6, G7) can improve the maximum rotation speed and exercise time that mice can withstand in the rotating cage. Among them, the G7 group has the strongest recovery of mice's exercise ability, almost returning to healthy levels ( Figure 10 E, F). The results of inflammatory factor levels showed that the G7 group had the strongest effect in reducing the levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α in the synovium and serum. Compared with the model group, the G6 group inhibited 54.3% of the abnormal secretion of inflammatory factors in the synovium, while the G7 group inhibited 78.8%. Similarly, the G6 group inhibited 63.7% of the abnormal secretion of inflammatory factors in the serum, while the G7 group inhibited 85.6% ( Figure 10 G, H). The results of spleen cell antigen restimulation showed that the IFN-γ level in group G7 was the lowest and the inhibitory effect was the strongest ( Figure 10 I).

[0151] Micro-CT was used to image the hind paws of each group of mice and observe bone erosion. Three-dimensional images of the paw bones show that all joints of RA mice (G2) showed bone erosion, especially the ankle and finger joints, with rough bone surfaces. The paw bones of healthy mice (G1) had normal morphology, smooth and intact surfaces. The ankle bones of the free adapter G3 and G4 groups were severely eroded, with partial loss of finger joint bones. The severity of bone erosion in the αFITC-CAR-T group was comparable to that of the RA group. The ankle joint structures of the G6 and G7 groups were normal, with smooth bone surfaces, and only some finger joints in the G6 group showed bone erosion ( Figure 10 J). Micro-CT images of the coronal sections of the ankle joints of mice showed that the trabecular bone density of the model group mice decreased and some of the parenchymal bone was lost, while the G6 and G7 groups could significantly restore the trabecular bone density of the tibia, fibula and talus, and protect the parenchymal bone ( Figure 10 J).

[0152] (2) Analysis of immune cells in synovium, lymph nodes, and spleen

[0153] The triple adapter combined with αFITC-CAR-T can significantly reduce B220 in synovium + B cells, CD4 + T cells and CD11b + / CD11c + The proportion and number of APCs in the G6 group were compared with those in the model group. + B cells, CD4 + T cells and CD11b + / CD11c + The number of APCs decreased by 37.1, 29.4 and 31.2% respectively; the effect was stronger in the G7 group, which decreased by 60.0, 51.0 and 52.6% respectively ( Figure 11A, B). The G7 group had the strongest effect in activating αFITC-CAR-T in the synovium, with the number of αFITC-CAR-T cells being 2.4 and 8.0 times that of the G5 and G6 groups, respectively ( Figure 11 A, B). Similarly, in lymph nodes and spleen, G7 group significantly reduced B220 + B cells, CD4 + T cells and CD11b + / CD11c + The proportion and number of APCs effectively promoted the proliferation of αFITC-CAR-T ( Figure 12 C, D).

[0154] (3) Safety evaluation

[0155] Biochemical analysis of the serum of mice in each group showed that there was no significant difference in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in each group of mice. Figure 12 Similarly, the results of routine blood analysis showed that the number of white blood cells (WBC), red blood cells (RBC), platelet count, mean platelet volume, mean corpuscular volume and red blood cell volume distribution width of each group of mice remained at normal levels ( Figure 12 ).

[0156] In summary, the triple adapter αCD4 / CTLA4 / αCD19-FITC combined with αFITC-CAR-T demonstrated significant efficacy in the treatment of CIA mouse models, effectively reducing RA scores and paw thickness, inhibiting the expression of inflammatory factors, restoring the mice's motor ability, and clearing target cells in the synovium, lymph nodes, and spleen. Furthermore, this therapeutic strategy demonstrated a high safety profile in vivo.

[0157] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An adapter hydrogel combined with modular CAR-T product, characterized in that: include: A liquid preparation containing an adapter, wherein the adapter contains an antibody or fusion protein modified with a marker molecule, wherein the antibody or fusion protein specifically recognizes a target cell surface marker; A liquid preparation containing a gel precursor solution containing a gel matrix for forming a temperature-sensitive gel; A liquid preparation containing CAR-T cells, wherein the chimeric antigen receptor in the CAR-T cells targets the marker molecule.

2. The adapter hydrogel combined with modular CAR-T product according to claim 1, characterized in that: Contains at least one of the following characteristics: (1) The gel matrix includes the copolymer polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid; (2) The labeling molecule includes a fluorescent molecule; (3) The antibody or fusion protein includes one or more of αCD4 antibody, αCD19 antibody, and CTLA4 Ig fusion protein; (4) In the adapter, the ratio of the number of the connected labeling molecule to the antibody or fusion protein is (0.5-5):

1.

3. The adapter hydrogel combined with modular CAR-T product according to claim 2, characterized in that: Contains at least one of the following characteristics: (1) The concentration of the gel matrix in the gel precursor solution is 15-30% (w / v); (2) The labeling molecule includes FITC.

4. The adapter hydrogel combined with modular CAR-T product according to claim 1, characterized in that: The CAR-T cells are obtained by transfecting T cells with chimeric antigen receptors and have at least one of the following characteristics: (1) The chimeric antigen receptor contains a coding sequence for a marker molecule antibody scFv; the marker molecule antibody scFv includes an αFITC scFv; (2) The chimeric antigen receptor contains a hinge region coding sequence, a transmembrane region coding sequence, and an intracellular signaling region coding sequence; (3) The chimeric antigen receptor contains a tag sequence.

5. The adapter hydrogel combined with modular CAR-T product according to claim 4, characterized in that: Contains at least one of the following characteristics: (1) The hinge region includes the CD28 hinge region; (2) the transmembrane region includes the CD28 transmembrane region; (3) The intracellular signaling region contains the costimulatory molecule region and the signal transduction molecule region; The co-stimulatory molecule region includes the CD28 intracellular signal region; the signal transduction molecule region includes the CD3zeta intracellular signal region.

6. A pharmaceutical composition, characterized in that contain: At least one antibody or fusion protein modified with a marker molecule, wherein the antibody or fusion protein specifically recognizes a target cell surface marker; the at least one antibody or fusion protein modified with a marker molecule includes an αCD4 antibody modified with a marker molecule, an αCD19 antibody modified with a marker molecule, and a CTLA4 Ig fusion protein modified with a marker molecule; A gel matrix, wherein the gel matrix is a thermosensitive gel matrix that forms a gel at body temperature; An immune cell containing a chimeric antigen receptor that targets the marker molecule.

7. The pharmaceutical composition according to claim 6, characterized in that The gel matrix includes a copolymer polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid; And / or, the immune cells include T cells.

8. Use of the adapter hydrogel combined with the modular CAR-T product according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 or 7 in the preparation of a pharmaceutical product, characterized in that: The pharmaceutical products include products for treating autoimmune diseases.

9. The use according to claim 8, characterized in that The pharmaceutical product is a product for treating rheumatoid arthritis.

10. A drug for treating rheumatoid arthritis, characterized in that: Comprising the adapter hydrogel combined with the modular CAR-T product according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 or 7.

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