Immunoregulation method for directional differentiation of CD4 + T cells

Abstract

The invention belongs to the technical field of biological medicines, and discloses an immunoregulation method for directional differentiation of CD4 + T cells. According to the method, an intelligent response type three-dimensional support composed of a temperature-sensitive block copolymer, MMP degradable peptide and a pH-sensitive component is constructed, a space-time nest is formed in situ at a focus, three active molecules including vorinostat, IL-6 / rapamycin and TGF-beta1 / retinoic acid are loaded by adopting a layered drug loading strategy, dynamic release is performed according to a microenvironment, and the pH-sensitive drug-loaded vorinostat / IL-6 / rapamycin / TGF-beta1 / retinoic acid is obtained. The efficient, lasting and antigen-specific directional induction of CD4 + T cells to subgroups such as Treg or Th17 is realized. The method is not only suitable for various disease scenes such as tumors, autoimmune diseases and chronic inflammation, but also can flexibly adapt to different T cell subpopulation induction requirements by replacing loaded bioactive molecule combinations and antigen peptide sequences, the local T cell regulation efficiency can be remarkably improved, systemic toxicity is avoided, and the method is suitable for clinical application. And good biocompatibility and clinical transformation potential are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a CD4 + Immunomodulatory methods for directed T cell differentiation. Background Technology

[0002] CD4 + As the core regulator of the adaptive immune response, the functional diversity of T cells is highly dependent on the precise guidance of their differentiation fate—in a specific microenvironment, initial CD4+... + T cells can differentiate into various effector or regulatory subsets such as Th1, Th2, Th17, and Treg, which respectively mediate anti-tumor immunity, allergic reactions, autoimmune inflammation, or immune tolerance.

[0003] In recent years, based on CD4 + Immunomodulatory strategies for targeted T cell differentiation have shown great potential in areas such as tumor immunotherapy, intervention for autoimmune diseases, and management of chronic inflammation. However, accurately constructing and sustainably maintaining a local signaling ecosystem conducive to the differentiation of target T cell subsets in a complex and dynamically changing in vivo environment at specific lesion sites (such as the microenvironment of solid tumors or the synovial tissue of rheumatoid arthritis) has always been a challenge.

[0004] Current technologies mostly rely on systemic administration to deliver cytokines (such as IL-2, TGF-β, IL-6, etc.), metabolic regulators, or small molecule epigenetic drugs in order to guide CD4. + T cells differentiate into the desired phenotype. Although such methods can detect elevated concentrations of target factors at the systemic level and have observed certain immunomodulatory effects in some models, their mechanisms of action inherently lack spatial specificity and temporal controllability.

[0005] Systemic exposure not only leads to a large amount of drug being distributed to non-target tissues, causing off-target toxicity (such as IL-2-induced vascular leakage syndrome), but more importantly, it makes it difficult to form a multi-factor synergistic signaling network with sufficient concentration gradient and duration at the lesion site—the latter being a core feature of the differentiation niche upon which T cell fate determination depends. Correspondingly, even local injection of a single factor cannot simulate the dynamic microenvironment constructed by stromal cells, antigen-presenting cells, and extracellular matrix under physiological conditions due to rapid clearance in vivo, enzymatic inactivation, and lack of scaffold support.

[0006] Traditional delivery systems cannot respond to pathophysiological signals specific to lesions (such as local acidosis, high expression of matrix metalloproteinases, or slight temperature rise) in passive diffusion carriers, thus failing to achieve on-demand release. At the same time, liquid formulations lacking three-dimensional structural support are difficult to form long-term biocompatible nests in the interstitial space, resulting in rapid diffusion of induction signals and inability to maintain the continuous stimulation required for differentiation. Summary of the Invention

[0007] To achieve the above-mentioned objectives, the present invention provides a CD4 + The core of the immunomodulatory method for directed T cell differentiation lies in constructing a three-dimensional biomaterial scaffold system with lesion microenvironment responsiveness. This system forms a spatiotemporal nest in situ within the target tissue, dynamically modulating the release spectrum of multiple factors. This precisely mimics the structure-signal-time synergistic characteristics of the natural T cell differentiation niche, thereby achieving targeted T cell differentiation. + Efficient, durable, and spatially defined targeted induction of T cells to specific effector or regulatory subsets.

[0008] The immune regulation method of the present invention includes the following steps: First, a biocompatible scaffold material composed of a smart responsive polymer network is prepared, which is in a liquid state or an injectable gel precursor state in vitro; second, at least two types of materials used to induce CD4 are... + Bioactive molecules for T cell-directed differentiation are loaded into the scaffold material. These bioactive molecules include, but are not limited to, cytokines, metabolic regulators, and epigenetic regulators. Subsequently, the scaffold material loaded with bioactive molecules is delivered to the target lesion site via local injection or minimally invasive implantation. Triggered by specific pathological signals in the lesion microenvironment, the scaffold material undergoes physical or chemical structural transformations, forming a stable three-dimensional porous network. The release rate and sequence of the loaded bioactive molecules are dynamically regulated according to local microenvironment parameters, thereby continuously constructing and maintaining a signaling ecosystem conducive to the differentiation of target T cell subsets at the lesion site.

[0009] The intelligent responsive polymer network is composed of thermosensitive block copolymers, matrix metalloproteinase (MMP) degradable peptide crosslinking units, and pH-sensitive ionic bonds. The basic framework of the polymer network is poly(N-isopropylacrylamide)-b-poly(ethylene glycol)-b-poly(N-isopropylacrylamide) triblock copolymer (PNIPAM-b-PEG-b-PNIPAM), with a low critical solution temperature (LCST) set at 39°C. This allows the material to remain in a sol state at body temperature (37°C), while rapidly undergoing hydrophobic aggregation under localized micro-heating conditions (≥39°C) common in inflammatory or tumor lesions, forming a physically crosslinked primary gel network. Building upon this, a MMP-2 / 9 specific substrate peptide composed of the sequence Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln was introduced as a chemical cross-linking point. Maleimide groups were modified at both ends of this peptide, which underwent Michael addition reactions with the thiol groups on the polymer chain, forming a covalent cross-link that could be cleaved by the highly expressed MMP-2 / 9 enzyme in the lesion. Simultaneously, carboxymethyl chitosan and arginine-functionalized hyaluronic acid were incorporated into the network. At physiological pH (7.4), both exhibit a loose conformation due to electrostatic repulsion. However, in the acidic microenvironment of the lesion (pH 6.2-6.8), the carboxyl grouping of carboxymethyl chitosan leads to charge shielding, promoting the formation of multiple hydrogen bonds and hydrophobic interactions between the arginine-functionalized hyaluronic acid and carboxymethyl chitosan, thereby enhancing the mechanical stability of the network and slowing down the molecular diffusion rate.

[0010] In a preferred embodiment of the present invention, the bioactive molecules are loaded into the scaffold material using a layered encapsulation strategy. The first type of bioactive molecule is a combination of TGF-β1 and retinoic acid, encapsulated within polylactic acid-glycolic acid copolymer nanospheres with a particle size of 200±50 nm. The polylactic acid-glycolic acid copolymer has a lactic acid:glycolic acid molar ratio of 75:25, a molecular weight of 45 kDa, and a degradation period of approximately 7 days. The second type of bioactive molecule is IL-6 and rapamycin, encapsulated within mesoporous silica nanoparticles with a particle size of 80±20 nm. The mesoporous silica nanoparticles have a pore size of 3.5 nm and are surface-modified with 3-aminopropyltriethoxysilane and grafted with a pH-sensitive hydrazone-linked PEG end-capping layer. The third type of bioactive molecule is the histone deacetylase inhibitor vorinostat, which is directly dissolved in the gel precursor solution in free form. The above three drug-loading units are uniformly dispersed in the scaffold precursor solution at a mass ratio of 1:1:0.5.

[0011] After the scaffold material is delivered to the lesion site, its release behavior exhibits a clear temporal sequence. In the initial stage (0-24 hours), due to the hydrophobic collapse of the PNIPAM segments triggered by local micro-heating, the scaffold rapidly gels. Simultaneously, the free vorinostatin, with its small molecular weight (264.32 Da) and lack of carrier binding, is preferentially and rapidly released via the Fickian diffusion mechanism, reaching a peak local concentration (approximately 1.2 μM) within 6 hours, acting on the initially infiltrated CD4+. + T cells enhance chromatin accessibility to Foxp3 and RORγt gene loci. During days 2-5, the MMP-2 / 9 enzymes highly expressed in the lesions cleave and degrade peptide cross-linking sites, leading to increased network porosity. Simultaneously, the acidic environment causes the PEG-capping layer on the surface of mesoporous silica nanoparticles to detach, exposing mesoporous channels and facilitating the simultaneous release of IL-6 and rapamycin. IL-6 concentrations are maintained at 20-50 ng / mL, and rapamycin concentrations at 5-10 nM. These two substances synergistically activate the STAT3 signaling pathway and inhibit mTORC1 activity, driving upregulation of RORγt expression. Between days 5 and 14, the polylactic acid-glycolic acid copolymer nanospheres rupture due to hydrolytic degradation, releasing TGF-β1 (concentration maintained at 5-10 ng / mL) and retinoic acid (concentration maintained at 100-200 nM). Together, they enhance Foxp3 expression and stabilize the Treg phenotype, or promote Th17 terminal differentiation in the presence of IL-6. Their specific fate depends on the intensity and duration of the previous IL-6 / rapamycin exposure.

[0012] The three-dimensional porous structure of the scaffold material described in this invention is prepared by freeze-drying process. Its pore size distribution is 50-200μm, porosity is 92%, and compression modulus is 8±2kPa. These mechanical properties match those of soft tissues (such as synovium or tumor stroma), and can effectively resist tissue compression and maintain long-term residence.

[0013] In another preferred embodiment of the present invention, the scaffold material further integrates an antigen-specific signaling module. Specifically, oxidized dextran is added to the gel precursor, and its aldehyde group reacts with the amino groups on the surface of liposomes loaded with major histocompatibility complex class II (MHCII) antigenic peptide tetramers via a Schiff base reaction, covalently anchoring the tetramers to the network backbone. The MHCII-antigenic peptide tetramers target specific disease-related antigens, such as the human leukocyte antigen DR4 (HLA-DR4) molecule binding to citrullinated fibrinogen peptide (Cit-Fib) in a rheumatoid arthritis model. 36-49 In a melanoma model, IA was used. b The molecule binds to the peptide of tyrosinase-associated protein 2 (TRP-2). 180-188 This design ensures that only the initial CD4... +T cells receive complete co-stimulatory and differentiation signals only when they recognize specific antigens presented on the scaffold through their T cell receptors, thereby achieving antigen-directed precise immune regulation and avoiding non-specific T cell activation.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. By constructing spatiotemporal nests in situ at the lesion using intelligent responsive biomaterial scaffolds, spatial confinement of differentiation induction signals is achieved, avoiding off-target toxicity caused by systemic drug administration; 2. By utilizing a multi-level drug delivery system and microenvironment response mechanism, the release kinetics of various bioactive molecules are precisely controlled, reproducing the temporal synergistic characteristics of signaling molecules in natural differentiation niches, and solving the signal antagonism problem caused by asynchronous factor release in traditional combination drug administration. 3. The three-dimensional porous structure of the scaffold provides physical support for long-term residence, maintaining a local microenvironment with high concentration of signals, which is significantly better than single-factor injection; 4. After integrating the antigen-specific presentation module, targeted regulation of disease-related T cell clones is achieved, improving the precision and safety of treatment; 5. All material components are known biocompatible substances, the preparation process complies with medical device manufacturing standards, and there is a clear clinical translation pathway.

[0015] 6. Overcoming the inherent limitations of traditional immune regulation strategies in terms of spatial precision, temporal controllability, and signal synergy, this study is the first to deeply integrate the scaffold design concept of tissue engineering with the differentiation niche theory of immunology, providing a method for achieving CD4 at the organ or lesion level. + A universal technology platform for T cell fate programming. This method is applicable not only to various disease scenarios such as tumors, autoimmune diseases, and chronic inflammation, but also to different T cell subset induction needs by changing the combination of loaded bioactive molecules and antigen peptide sequences, showing broad application prospects and industrialization value. Detailed Implementation

[0016] This invention provides a CD4 + An immunomodulatory method for directed T cell differentiation relies on a lesion-microenvironment-responsive three-dimensional biomaterial scaffold system. This system constructs a spatiotemporal nest with dynamic multi-factor release capabilities in situ within the target tissue, precisely replicating the structure-signal-time tripartite regulatory logic upon which the natural T cell differentiation niche depends. This method achieves targeted T cell differentiation through local delivery, intelligent response, and sequential release mechanisms. + Efficient, durable, and spatially defined targeted induction of T cells to specific effector or regulatory subsets.

[0017] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0018] Example 1: The scaffold contains a thermosensitive copolymer + MMP degradable peptide + pH-sensitive ionic bonds; it is layered with TGF-β1 / retinoic acid (polylactic acid-glycolic acid copolymer microspheres), IL-6 / rapamycin, and vorinostat (free); the mass ratio is 1:1:0.5; the delivery method is intrasynovial injection; the antigen module is HLA-DR4-Cit-Fib. 36-49 ; Control process: Stent fabrication → Layered drug loading → Ultrasound-guided local injection → Gelation triggered by lesion microenvironment → Sequential release of active molecules → Targeted induction of CD4 + T-cell differentiation → Effect detection.

[0019] Example 2: Drug loading to enhance the IL-6 / rapamycin ratio, antigen module IA b -TRP-2 180-188 Delivery method: intratumoral injection; other formulations and processes are the same as in Example 1; Control process: Same as Example 1 (adjustment of drug loading ratio + replacement of antigen module).

[0020] Example 3: Removal of MHCII-antigen peptide tetramer liposomes; the remaining formulation and process are the same as in Example 1; Control process: Same as in Example 1 (antigen module integration steps).

[0021] Example 4: The content of MMP degradable peptide crosslinking units was increased by 50%; the rest of the formulation and process were the same as in Example 1; Control process: Same as in Example 1 (adjustment of stent crosslinking unit ratio).

[0022] Example 5: The degree of substitution of carboxymethyl chitosan was increased to 0.9; the remaining formulation and process were the same as in Example 1; Control procedure: Same as in Example 1 (adjustment of pH-sensitive components of the stent).

[0023] Example 6: Polylactic acid-glycolic acid copolymer microspheres: mesoporous silica nanoparticles: vorinostat = 1:1:0.8; other formulations and processes are the same as in Example 1; Control procedure: Same as Example 1 (adjustment of drug loading ratio).

[0024] Example 7: Delivery method is endoscopic-assisted minimally invasive implantation; the rest of the formula and process are the same as in Example 1; Control process: Same as Example 1 (with adjustment of delivery method).

[0025] Example 8: The thermosensitive copolymer has a lower critical solution temperature of 38°C; the remaining formulation and process are the same as in Example 1; Control process: Same as in Example 1 (adjustment of thermosensitive copolymer properties).

[0026] Comparative Example 1: No smart scaffold, active molecules were injected systemically at the same dose; other test conditions were the same as in Example 1; Control process: Mixing of active molecules → Intravenous injection → Systemic distribution → Effect detection.

[0027] Comparative Example 2: The scaffold has no responsive properties (only thermosensitive copolymer), and all active molecules are mixed and loaded without stratification; the rest of the formulation and process are the same as in Example 1; Control process: standard stent preparation → drug loading → local injection → efficacy testing.

[0028] Test method: Differentiation efficiency test: Flow cytometry detection of the target subset's CD4 content + T cell ratio; immunohistochemical analysis of local immune cell infiltration in lesions.

[0029] Disease intervention tests: Arthritis model to detect clinical scores and bone erosion area; tumor model to detect tumor inhibition rate and degree of necrosis.

[0030] Safety and targeting testing: detecting liver and kidney function and systemic inflammatory markers; assessing the degree of non-specific immune activation; monitoring stent residence time and degradation.

[0031] The test data comparisons are shown in Table 1 and Table 2.

[0032] Table 1. Comparison of target subgroup proportions, disease intervention effects, and tissue protection / tumor suppression effects. ; Table 2 Comparison of stent residence time and systemic toxicity ; Examples 1-8 showed that the target subgroup accounted for ≥36.8% and the disease intervention effect was ≥58%, which was far superior to the control examples. Control example 1 showed poor targeting and significant toxicity of systemic drug delivery, while control example 2 showed low differentiation efficiency due to disordered release. This demonstrates that the smart scaffold + layered sequential release is the key to precise regulation.

[0033] The antigen module (Example 1 vs Example 3) improved the targeted differentiation efficiency by 41%; the response characteristics were enhanced (Examples 4 and 5) to further optimize the intervention effect; and the drug loading ratio was adjusted (Example 6) to enhance signal synergy.

[0034] The embodiment demonstrates local regulation with no significant systemic toxicity, making it suitable for different disease models; the stent has a long residence time, eliminating the need for repeated drug administration; the antigen module enables precise targeting, avoiding non-specific immune activation.

[0035] Compared to systemic drug delivery (Comparative Example 1), the proportion of Tregs in the example increased by 213%, and the disease intervention effect increased by 244%. Compared to ordinary disordered drug delivery (Comparative Example 2), the differentiation efficiency increased by 109%, solving the industry problem of poor targeting and insufficient signal synergy in traditional immune regulation.

[0036] In summary, the method described in this invention, through the synergy of a smart scaffold and layered drug delivery, can achieve CD4 activation with different parameter combinations. + T cells can differentiate efficiently and in a targeted manner, making them suitable for scenarios such as autoimmune diseases and tumors, and have good potential for clinical translation.

[0037] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A CD4 + An immunomodulatory method for directed T cell differentiation, characterized in that, Includes the following steps: Prepare a biocompatible scaffold material composed of a smart responsive polymer network, wherein the scaffold material is in a liquid state or an injectable gel precursor state in vitro; At least two are used to induce CD4 + Bioactive molecules for T cell directed differentiation are loaded into the scaffold material; The scaffold material loaded with the bioactive molecules is delivered to the target lesion site via local injection or minimally invasive implantation. Triggered by specific pathological signals in the lesion microenvironment, the scaffold material undergoes physical or chemical structural transformation, forming a stable three-dimensional porous network. Based on local microenvironment parameters, the release rate and release order of the loaded bioactive molecules are dynamically regulated, thereby continuously constructing and maintaining a signaling ecosystem conducive to the differentiation of target T cell subsets in the lesion.

2. The CD4 according to claim 1 + An immunomodulatory method for directed T cell differentiation, characterized in that, The intelligent responsive polymer network is composed of a thermosensitive block copolymer, MMP degradable peptide crosslinking units, and pH-sensitive ionic bonds. The thermosensitive block copolymer is a poly(N-isopropylacrylamide)-b-poly(ethylene glycol)-b-poly(N-isopropylacrylamide) triblock copolymer with a lower critical dissolution temperature of 39°C.

3. The CD4 according to claim 2 + An immunomodulatory method for directed T cell differentiation, characterized in that, The MMP degradable peptide crosslinking unit is an MMP-2 / 9 specific substrate peptide composed of the sequence Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, which is modified at both ends by maleimide groups and crosslinked with thiol groups on the polymer chain.

4. The CD4 according to claim 2 + An immunomodulatory method for directed T cell differentiation, characterized in that, The pH-sensitive ionic bonds are formed by carboxymethyl chitosan and arginine-functionalized hyaluronic acid under acidic conditions.

5. The CD4 according to claim 1 + An immunomodulatory method for directed T cell differentiation, characterized in that, The bioactive molecules are loaded using a layered encapsulation strategy, including: The first type of bioactive molecule is a combination of TGF-β1 and retinoic acid, encapsulated in polylactic acid-glycolic acid copolymer nanospheres, wherein the polylactic acid-glycolic acid copolymer has a lactic acid:glycolic acid molar ratio of 75:25 and a molecular weight of 45kDa. The second type of bioactive molecules are IL-6 and rapamycin, which are encapsulated in mesoporous silica nanoparticles. The surface of the mesoporous silica nanoparticles is modified with 3-aminopropyltriethoxysilane and then grafted with a pH-sensitive hydrazone-linked PEG end-capping layer. The third type of bioactive molecule is vorinostat, which is dissolved directly in the gel precursor solution in its free form.

6. The CD4 according to claim 5 + An immunomodulatory method for directed T cell differentiation, characterized in that, The polylactic acid-glycolic acid copolymer nanospheres, mesoporous silica nanoparticles, and vorinostat are uniformly dispersed in the scaffold precursor solution at a mass ratio of 1:1:0.

5.

7. The CD4 according to claim 1 + An immunomodulatory method for directed T cell differentiation, characterized in that, Under local micro-heating conditions at the lesion site, the scaffold material rapidly gels, and free vorinostat is preferentially released within 0-24 hours, with a local concentration reaching 1.2 μM within 6 hours; On days 2-5, the MMP-2 / 9 enzyme highly expressed in the lesions cleaves and degrades the peptide cross-linking units. At the same time, the acidic microenvironment promotes the shedding of the PEG end capping layer on the surface of the mesoporous silica nanoparticles, releasing IL-6 and rapamycin simultaneously. On days 5-14, the polylactic acid-glycolic acid copolymer nanospheres hydrolyze and degrade to release TGF-β1 and retinoic acid.

8. The CD4 according to claim 1 + An immunomodulatory method for directed T cell differentiation, characterized in that, The scaffold material is freeze-dried to form a three-dimensional porous structure.

9. The CD4 according to claim 8 + An immunomodulatory method for directed T cell differentiation, characterized in that, The mechanical properties of the three-dimensional porous structure are matched with those of synovial tissue or tumor stroma, which are used to resist tissue compression and maintain the scaffold at the lesion site for more than 14 days.

10. The CD4 according to claim 1 + An immunomodulatory method for directed T cell differentiation, characterized in that, The scaffold material further integrates an antigen-specific signaling module, which includes oxidized dextran and liposomes loaded with MHCII-antigen peptide tetramers, wherein the aldehyde groups of the dextran and the amino groups on the surface of the liposomes are covalently anchored to the polymer network backbone via a Schiff base reaction.