A chimeric antigen receptor dendritic cell product
By constructing and delivering CAR-DCs in dendritic cells, the problems of tumor microenvironment inhibition and antigenic heterogeneity in solid tumors by CAR-T therapy have been solved, achieving highly efficient treatment of solid tumors and showing good clinical application prospects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
- Filing Date
- 2026-04-16
- Publication Date
- 2026-07-14
Smart Images
Figure CN122382011A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the generation of a chimeric antigen receptor dendritic cell product and its application in tumor immunotherapy. Background Technology
[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.
[0003] Chimeric antigen receptor-modified T-cell (CAR-T) therapy, a type of immunotherapy, has shown significant clinical efficacy and is one of the focal points and hot topics in cancer immunotherapy research. Clinical practice has confirmed that while CAR-T cells exert their anti-tumor effects, they release a large number of cytokines, playing a double-edged sword role: on the one hand, they rely on secreted cytotoxic granzymes and perforin to destroy tumor cells; on the other hand, the cytokine "storm" poses safety risks: CAR-T cell therapy can induce T cells, B cells, and NK cells to release large amounts of inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, triggering an acute inflammatory response that induces epithelial and tissue damage, leading to microvascular leakage and causing nausea, headache, tachycardia, shortness of breath, and even other potentially fatal clinical reactions.
[0004] Currently, CAR-T therapy shows clear efficacy against hematological malignancies (such as leukemia and lymphoma), but its efficacy against solid tumors is poor. The core reasons include: 1. Tumor microenvironment (TME) suppression: Solid tumors are surrounded by immunosuppressive cells (such as Tregs and M2 macrophages) and inhibitory cytokines (such as TGF-β and IL-10), which inhibit the activation, proliferation, and killing ability of CAR-T cells. 2. Antigen heterogeneity and loss: Solid tumor antigen expression is unstable (some tumor cells do not express target antigens), causing CAR-T cells to be unable to recognize all tumor cells, easily leading to relapse; some tumors can also evade attack by "losing target antigens." 3. Difficulty in tumor invasion: Solid tumors have a dense stromal barrier (such as collagen), making it difficult for CAR-T cells to penetrate and reach the tumor core to exert their effects. Traditional CAR cell therapy involves the in vitro modification of autologous cells, a cumbersome and expensive process that poses serious challenges to its applicability, patient accessibility, and quality control. In vivo CAR cell manufacturing offers a promising solution to these challenges. The ability of lipid nanoparticles (LNPs) to facilitate gene delivery offers the potential for manufacturing CAR cells in vivo. Summary of the Invention
[0005] To address the shortcomings of existing technologies, and given the crucial role of dendritic cells (DCs) in anti-tumor immunity, this invention constructed and evaluated CAR-DCs to explore their therapeutic potential in solid tumors. Results showed that, unlike CAR-T cells, CAR-DCs can initiate a broader range of antigen-specific immune responses (not limited to the target antigens of CARs), which helps overcome the heterogeneity of solid tumors. Furthermore, CAR-DCs can enhance the infiltration of immune cells into tumors, reshaping the tumor immunosuppressive microenvironment.
[0006] Antigen heterogeneity and the immunosuppressive microenvironment have been key factors limiting the successful application of CAR-T therapy in solid tumors. To overcome these limitations, CAR cell therapy has expanded from T cells to a variety of other immune cells. Considering the central role of dendritic cells (DCs) in anti-tumor immunity, this invention designs and constructs CAR-DCs to rapidly and efficiently stimulate anti-tumor immune responses and effectively inhibit tumor growth. Unlike CAR-T cells, which are limited to specific targets, T cells activated by CAR-DCs through phagocytosis of tumor antigens can act on multiple antigens, exhibiting functions similar to whole-cell vaccines. This characteristic helps overcome the challenge of solid tumor heterogeneity. Furthermore, CAR-DCs can enhance the infiltration of immune cells into tumors and reshape the immunosuppressive microenvironment. In conclusion, CAR-DCs bring new possibilities to the treatment of solid tumors.
[0007] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a chimeric antigen receptor dendritic cell product is provided, characterized in that it comprises dendritic cells containing a chimeric antigen receptor (CAR), said CAR being composed of the following: Antigen recognition region (scFv): The tumor antigens targeted by the scFv are CEA, HER2, EGFR, CD19, CD20, BCMA, CD22, CD30, CD33, MSLN, PSMA, GPC3, PSA, PD-L1, etc. Hinge region: Selected from the hinge regions of CD8, CD8α, CD28, IgG, IgG1, IgG4 or their functional variants; Transmembrane region: selected from the transmembrane regions of CD8, CD8α, CD3ζ, CD4, CD28 or their functional variants; Intracellular region: Selected from one or more of CD3ζ, FcγR, 4-1BB, CD28, OX40, ICOS, CD27 or their functional variants.
[0008] In a second aspect of the invention, a chimeric antigen receptor (CAR) expression system for selective expression in dendritic cells is provided, comprising a nucleic acid molecule capable of expressing the CAR in dendritic cells (DCs), wherein the sequence encoding the CAR sequentially comprises sequences encoding the following elements: a single-stranded variable fragment (scFv), a hinge region, a transmembrane region, and an intracellular signal transduction domain.
[0009] Furthermore, the expression system comprises a nucleic acid molecule capable of expressing CAR in dendritic cells (DCs), the nucleic acid molecule comprising, from the 5' end to the 3' end, the following operatively linked genetic elements: a dendritic cell-specific promoter, a sequence encoding a chimeric antigen receptor (CAR); wherein the sequence encoding the CAR comprises, in turn, sequences encoding the following elements: a signal peptide, a single-stranded variable fragment (scFv), a hinge region, a transmembrane region, and an intracellular signal transduction domain.
[0010] In one or more embodiments of the present invention, the dendritic cell-specific promoter includes, but is not limited to, the ZBTB46 promoter, the CD209 promoter, the CD11c promoter, BDCA-2 (CD303), or functional variants thereof. The ZBTB46 promoter gene origin is the human (or mouse) ZBTB46 gene, which is primarily highly expressed on the surface of dendritic cells (DCs). The sequence origin is the regulatory region upstream of the transcription start site of the ZBTB46 gene, containing core promoter elements and adjacent regulatory sequences that enhance its dendritic cell specificity. Its core function is to initiate the transcription of downstream genes.
[0011] This invention utilizes the dendritic cell specificity of the ZBTB46 promoter to restrict CAR gene expression to the dendritic cell interior or a specific subset thereof. This achieves "targeted" editing: avoiding CAR expression in other immune cells such as T cells and NK cells, thereby precisely modifying dendritic cells to give them the ability to target tumor antigens, avoiding off-target effects or safety issues caused by gene editing of other cells.
[0012] In one or more embodiments of the present invention, the signal peptide is selected from the CD8α signal peptide or a functional variant thereof; protein source: human (or mouse, such as mCD8α) CD8α chain. CD8 is a co-receptor on the surface of T cells, composed of CD8α and CD8β chains; sequence location: located at the N-terminus of the CD8α protein, and is the first part translated during the synthesis of nascent proteins. Function: guides newly synthesized proteins into the secretory pathway, ultimately localizing them to the cell membrane.
[0013] In one or more embodiments of the present invention, the single-chain variable fragment scFv targets tumor-associated antigens. Source: Antibody source: The scFv is derived from a monoclonal antibody capable of specifically recognizing a certain tumor-associated antigen. Function: Responsible for specifically recognizing tumor antigens. The tumor-associated antigens targeted by the scFv include, but are not limited to, CEA, HER2, EGFR, CD19, CD20, BCMA, CD22, CD30, CD33, MSLN, PSMA, GPC3, PSA, PD-L1, etc. In one or more embodiments of the present invention, the single-chain variable fragment scFv is M5A, HER2, EGFR, CD19, CD20, or BCMA.
[0014] In one or more embodiments of the invention, the hinge region is selected from the hinge regions of CD8, CD8α, CD28, IgG, IgG1, IgG4, or functional variants thereof; Hinge region origin: Protein origin: Similar to the signal peptide, it is typically derived from the extracellular region of the CD8, CD8α, CD28, IgG, IgG1, or IgG4 chain, located between the immunoglobulin-like variable region and the transmembrane region. Function: Provides flexibility, allowing the scFv to overcome steric hindrance and contact the antigen.
[0015] In one or more embodiments of the present invention, the transmembrane region is selected from the transmembrane regions of CD8, CD8α, CD3ζ, CD4, CD28, or functional variants thereof; Transmembrane region source: Protein source: Transmembrane region selected from CD8, CD8α, CD3ζ, CD4, or CD28 chains, which is a hydrophobic amino acid sequence capable of embedding in the phospholipid bilayer of the cell membrane. Function: Anchoring protein: Stably anchors the CAR protein to the cell membrane of dendritic cells, becoming a membrane protein; Stabilizing structure: Together with the hinge region and intracellular domain, it maintains the overall spatial structure of the CAR.
[0016] In one or more embodiments of the present invention, the intracellular signal transduction domain is selected from one or more combinations of CD3ζ, FcγR, 4-1BB, CD28, OX40, ICOS, CD27, or functional variants thereof; CD3ζ origin: protein source: the ζ chain of the human (or mouse) T cell receptor CD3 complex. In native T cells, after the TCR recognizes an antigen, the CD3ζ chain is responsible for transmitting the initial activation signal. Function: Transmitting activation signal: This is the "switch" for CAR. When extracellular scFv binds to an antigen, it causes CAR molecule clusters, thereby activating the CD3ζ chain. Initiating cellular response: The CD3ζ chain contains immune receptor tyrosine based on the activation motif, and its phosphorylation initiates downstream signaling cascade reactions, ultimately triggering the activation of dendritic cells and the exercise of their immune functions (such as antigen presentation, cytokine secretion, etc.).
[0017] The nucleotide sequences of each element in this invention are not limited to the sequences listed in the examples, but also include variants, alleles, codon-optimized sequences, etc. that have high sequence identity (e.g., 80%, 85%, 90%, 95%, 99% or more) and retain the same function.
[0018] In one or more embodiments of the present invention, the nucleic acid molecule is in the form of DNA or RNA.
[0019] In a third aspect of the invention, a dendritic cell-targeting CAR vector is provided, comprising the CAR expression system described above, wherein the vector is a plasmid, a lentiviral vector, an adeno-associated virus vector, or an in vitro transcribed mRNA.
[0020] In one or more embodiments of the present invention, the backbone vector used for the plasmid includes, but is not limited to, PB[Exp]-Backbone.
[0021] In a fourth aspect of the invention, a dendritic cell-targeting lipid nanoparticle (LNP) delivery vector is provided, comprising the CAR expression system in the form of plasmid DNA; and an LNP encapsulating the plasmid DNA.
[0022] In a fifth aspect of the present invention, a method for preparing chimeric antigen receptor dendritic cells (CAR-DC) is provided, comprising introducing the CAR expression system or the CAR vector into dendritic cells (DC) to express the chimeric antigen receptor in the DC; the introduction is performed in vitro or in vivo.
[0023] In one or more embodiments of the present invention, the CAR-DC can initiate an immune response against a variety of tumor antigens, not limited to the antigen targeted by the CAR; and / or, the CAR-DC can enhance the infiltration of immune cells into tumor tissues and remodel the tumor immunosuppressive microenvironment.
[0024] In a sixth aspect of the invention, the use of the CAR expression system, the CAR vector, or the CAR-DC prepared by the method in the preparation of a medicament for the treatment or prevention of tumors, including solid tumors or hematologic malignancies.
[0025] In one or more embodiments of the present invention, the solid tumors include, but are not limited to, colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, stomach cancer, liver cancer, glioma, and prostate cancer; the hematologic tumors include, but are not limited to, leukemia, lymphoma, and multiple myeloma.
[0026] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising the CAR expression system, the CAR vector, or a CAR-DC prepared by the method, and a pharmaceutically acceptable vector.
[0027] In one or more embodiments of the present invention, the dosage form of the pharmaceutical composition is lipid nanoparticles (LNPs), wherein the LNPs encapsulate the CAR expression system or CAR carrier; the LNPs are prepared by the following method: (1) Preparation of lipid phase in lipid nanoparticles: Distearate phosphatidylcholine (DSPC), polyethylene glycol 2000-dimyristoylglycerol (PEG2000-DMG), ionizable cationic lipid SM-102, and cholesterol were dissolved in an organic solvent (preferably ethanol) to obtain a lipid phase solution; (2) Preparation of aqueous phase in lipid nanoparticles: The CAR expression system or the CAR vector is added to an acidic buffer solution to obtain an aqueous solution; (3) LNPs were prepared using microfluidic mixing.
[0028] Preferably, the ratio of DSPC, PEG2000-DMG, SM-102, cholesterol and organic solvent is (2~4) mg: (1~2) mg: (10~15) μl: (4~6) mg: (1~3) mL.
[0029] Preferably, the acidic buffer solution is a citrate buffer solution with a pH of 3.0.
[0030] Preferably, the ratio of the CAR carrier to the aqueous solution is (50~150) μg: (0.2~1) ml.
[0031] Preferably, the volume ratio of the lipid phase solution to the aqueous phase solution is 1:(2~4).
[0032] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: This invention constructs a chimeric antigen receptor dendritic cell product and a dendritic cell (DC)-targeting CAR expression system, and innovatively employs lipid nanoparticles (LNPs) for in vitro or in vivo delivery. The aim is to directly and specifically generate DCs expressing chimeric antigen receptors (CARs) in vitro or in vivo, thereby efficiently activating anti-tumor immune responses. This strategy exhibits high specificity, high efficiency, safety, and promising clinical translation prospects. Attached Figure Description
[0033] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0034] Figure 1 Schematic diagram of the structure of CAR and CARΔ plasmids.
[0035] Figure 2 Characterization diagrams of LNP@CAR and LNP@CARΔ in Example 2, where a represents the particle size distribution and morphology of LNP@CARΔ; b represents the particle size distribution and morphology of LNP@CAR; and c represents the gel electrophoresis diagram.
[0036] Figure 3 Percentage of EGFP-positive cells analyzed by flow cytometry under different treatments; A is a flow cytometry analysis graph; B is a bar chart of the percentage of EGFP-positive cells analyzed by flow cytometry.
[0037] Figure 4 Percentage of CTV-positive cells in DCs under different treatments; A is a graph showing the percentage of CTV-positive cells; B is a bar chart showing the percentage of CTV-positive cells.
[0038] Figure 5 Percentage of H-2Kb-SIINFEKL+ cells in CD11c+ under different treatments; A is a graph showing the percentage of H-2Kb-SIINFEKL+ cells in CD11c+; B is a bar chart showing the percentage of H-2Kb-SIINFEKL+ cells in CD11c+.
[0039] Figure 6 Subcutaneous colon cancer tumor volume (A) and tumor weight (B) under different treatments.
[0040] Figure 7 CAR structure diagram (A) and subcutaneous breast cancer tumor volume (B) and tumor weight (C) under different treatments.
[0041] Figure 8 CAR structure diagram (A) and subcutaneous lung cancer tumor volume (B) and tumor weight (C) under different treatments.
[0042] Figure 9 CAR structural schematic diagram (A), quantitative fluorescence images of hematologic malignancies under different treatments (B), and mouse survival time (C).
[0043] Figure 10 CAR structure diagram (A), subcutaneous colon cancer tumor volume changes (B), final volume bar chart (C), and tumor weight (D). Detailed Implementation
[0044] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0045] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.
[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0047] The nucleotide sequence used in this embodiment is: ZBTB46 promoter, whose nucleotide sequence is shown in SEQ.ID.NO.1; CD8α-leader, whose nucleotide sequence is shown in SEQ.ID.NO.2; M5A_scFv, whose nucleotide sequence is shown in SEQ.ID.NO.3; CD8 hinge, whose nucleotide sequence is shown in SEQ.ID.NO.4; CD8™, whose nucleotide sequence is shown in SEQ.ID.NO.5; CD3ζ, whose nucleotide sequence is shown in SEQ ID NO. 6; P2A, whose nucleotide sequence is shown in SEQ.ID.NO.7; Myc, whose nucleotide sequence is shown in SEQ.ID.NO.8; EGFP, whose nucleotide sequence is shown in SEQ.ID.NO.9.
[0048] HER2-scFv, whose nucleotide sequence is shown in SEQ.ID.NO.10; EGFR-scFv, whose nucleotide sequence is shown in SEQ.ID.NO.11; CD19-scFv, whose nucleotide sequence is shown in SEQ.ID.NO.12; CD20-scFv, whose nucleotide sequence is shown in SEQ.ID.NO.13; BCMA-scFv, whose nucleotide sequence is shown in SEQ.ID.NO.14; FcγR, whose nucleotide sequence is shown in SEQ.ID.NO.15; 4-1BB, whose nucleotide sequence is shown in SEQ.ID.NO.16; CD28, whose nucleotide sequence is shown in SEQ.ID.NO.17; OX40, whose nucleotide sequence is shown in SEQ.ID.NO.18; CD4 TM, whose nucleotide sequence is shown in SEQ.ID.NO.19; IgG hinge, whose nucleotide sequence is shown in SEQ.ID.NO.20.
[0049] Example 1 It should be noted that all single-chain variable fragments (scFv) used in the embodiments of this invention were designed and screened for human tumor-associated antigens and are capable of specifically recognizing human CEA, HER2, EGFR, CD19, CD20, and BCMA antigens. To perform in vivo functional validation in mouse models, this invention has correspondingly constructed mouse tumor cell lines that stably express the above-mentioned human antigens (such as CT26-hCEA, 4T1-hHER2, LLC-hEGFR, A20-hCD19, etc.).
[0050] like Figure 1 The preparation of a CAR plasmid includes the following steps: Preparation of promoter fragment: A DNA fragment containing the ZBTB46 promoter sequence was directly chemically synthesized and named pUp-ZBTB46 for later use.
[0051] Construction and validation of intermediate vectors for CAR expression units: Assembly: Using the Golden Gate cloning method, DNA fragments encoding the following elements were assembled sequentially: mouse CD8α signal peptide sequence, Myc tag sequence, M5A single-chain antibody variable region fragment, mouse CD8 hinge region sequence, mouse CD8 transmembrane region sequence, mouse CD3ζ intracellular signal domain, P2A self-cleaving peptide sequence, and enhanced green fluorescent protein reporter gene; this expression cassette was cloned into an intermediate vector to construct pDown-mCD8α-leader / Myc / M5A_scFv / mCD8-hinge / mCD8-TM / mCD3zeta:P2A:EGFP.
[0052] Verification: The obtained intermediate vector was subjected to Sanger DNA sequencing to confirm that the nucleotide sequence of the CAR expression unit was correct.
[0053] Final assembly of the CAR plasmid: The obtained pUp-ZBTB46 promoter fragment, the validated CAR expression unit intermediate vector, and the PB[Exp] backbone vector were recombined using the LR reaction method.
[0054] The target plasmid was successfully constructed: pPB[Exp]-ZBTB46>mCD8α-leader / Myc / M5A_scFv / mCD8-hinge / mCD8-TM / mCD3ζ:P2A:EGFP vector.
[0055] The preparation of a CARΔ plasmid involves first directly synthesizing pUp-ZBTB46 (promoter) chemically, and then constructing an intermediate vector pDown-mCD8α-leader / Myc / M5A_scFv / mCD8-hinge / mCD8-TM:P2A:EGFP carrying the target gene using the GoldenGate method. The sequence was verified to be correct by Sanger sequencing. Then, the pUp-ZBTB46 vector carrying the promoter, pDown-mCD8-leader / Myc / M5A_scFv / mCD8-hinge / mCD8-TM:P2A:EGFP, and PB[Exp]-Backbone were recombined using the LR reaction method to construct the final pPB[Exp]-ZBTB46>mCD8α-leader / Myc / M5A_scFv / mCD8-hinge / mCD8-TM:P2A:EGFP vector.
[0056] Example 2 The preparation of a pCAR-lipid nanoparticle includes the following steps: (1) Preparation of lipid phase in lipid nanoparticles: 2.75 mg DSPC, 1.15 mg PEG2000-DMG, 11.6 μl SM-102 and 4.7 mg cholesterol were dissolved in 1.66 ml anhydrous ethanol and the lipids were completely dissolved by vortex sonication.
[0057] (2) Preparation of aqueous phase in lipid nanoparticles: 100 μg CAR plasmid was added to sodium citrate solution at pH = 3 to 0.5 ml.
[0058] (3) Preparation using microfluidic mixing: The lipid phase solution and the aqueous phase solution were filtered through 0.22 μm filter membranes respectively. The aqueous and lipid phase solutions were drawn into corresponding syringes at a volume ratio of 3:1 and fixed to microinjection pumps. The syringes were connected to the inlet of the microfluidic chip via catheters. The flow rates of the two microinjection pumps were set: the aqueous phase flow rate was set to 1.5 mL / min, and the lipid phase flow rate was set to 0.5 mL / min. Before starting the microinjection pumps, the outlet of the microfluidic chip was connected to a catheter, and the end of the catheter was placed in an EP tube of appropriate volume. The mixed liquid flowing out of the chip outlet was collected as pCAR-lipid nanoparticles (denoted as LNP@CAR), i.e., the nanocarrier prepared in vitro.
[0059] The preparation of LNP@CARΔ is described above.
[0060] The obtained LNP@CAR was purified by dialysis. After purification, its morphology was observed and particle size was measured under a transmission electron microscope (JEOL). The encapsulation of the plasmid was verified by gel electrophoresis. In the presence of FBS and DNase I, LNP@CAR was not degraded, while pCAR (free CAR plasmid) was fully degraded, indicating that LNP successfully encapsulated the CAR, thus preventing plasmid degradation. Therefore, LNP@CAR exhibited higher stability than pCAR, demonstrating that LNPs can effectively encapsulate CAR plasmids. Figure 2 Dynamic light scattering analysis and transmission electron microscopy revealed that the sizes of LNP@CAR and LNP@CARΔ were 67.35±3.45 nm and 65.78±2.90 nm, respectively, both exhibiting spherical morphologies. Figure 2 a and b.
[0061] LNP@CAR-mediated in vitro transfection assay of dendritic cells
[0062] For in vitro gene transfection, BMDCs (hereinafter referred to as DCs), CT26 cells, and RAW264.7 cells were incubated in 12-well plates with physiological saline, pCAR△, pCAR, LNP@CAR△, or LNP@CAR at a plasmid dose of 30 μg / well (except for physiological saline). After 48 hours of incubation, the percentage of EGFP-positive cells was analyzed by flow cytometry.
[0063] The results are as follows Figure 3EGFP expression was low in both RAW264.7 and CT26 cells treated with any of the formulations. However, the EGFP positivity rate of DCs treated with LNP@CARΔ and LNP@CAR was significantly higher than that of RAW264.7 and CT26 cells, indicating that LNP@CARΔ and LNP@CAR can be specifically expressed in DCs. Furthermore, the EGFP positivity rate of the LNP@CARΔ and LNP@CAR groups was significantly higher than that of the pCARΔ and pCAR groups, indicating that LNPs can effectively protect plasmids and improve plasmid transfection efficiency. Moreover, there was no difference in the percentage of EGFP-positive cells between the LNP@CARΔ and LNP@CAR groups, suggesting that the presence of the CD3ζ gene does not affect plasmid transfection efficiency.
[0064] LNP@CAR-mediated dendritic cell phagocytic specificity and antigen presentation capacity
[0065] To assess their tumor phagocytic capacity, dendritic cells (DCs) were first incubated with saline, pCAR△, pCAR, LNP@CAR△, or LNP@CAR at a plasmid dose of 30 μg / well (except for saline) for 48 hours. CT26-CEA-OVA cells were labeled with CellTrace Violet (CTV) proliferation dye. Transfected DCs and CTV-labeled CT26-CEA-OVA cells were added to new 12-well plates at a 1:1 volume ratio. After incubation at 37°C and 5% CO2 for 5 hours, the CTV fluorescence intensity of DCs was detected.
[0066] The results are as follows Figure 4 The percentage of CTV-positive DCs in the LNP@CARΔ and LNP@CAR groups was higher than in other groups, with the LNP@CAR group showing the highest percentage. This result indicates that CAR-DCs produced by LNP@CAR can effectively internalize antigens from tumor cells or tumor cell fragments. Furthermore, compared to the saline and pCAR groups, the LNP@CARΔ group had a higher proportion of CTV-positive DCs, attributed to the addition of CARΔ to DCs via LNP@CARΔ treatment. Despite lacking an intracellular signaling domain, CARΔ retained its tumor-targeting ability, thereby inducing stronger phagocytosis against tumor cells or tumor cell fragments.
[0067] LNP@CAR-mediated dendritic cell antigen presentation capability
[0068] DCs from each formulation were cultured in 12-well plates. Cells were initially co-incubated with physiological saline, pCAR△, pCAR, LNP@CAR△, or LNP@CAR at a plasmid dosage of 30 μg / well (except for physiological saline). After 48 h of incubation, CT26-CEA-OVA cells were digested, and transfected DCs and CT26-CEA-OVA cells were added to new 12-well plates at a 1:1 volume ratio and incubated for 5 h. After co-incubation, cells were collected, washed with PBS, and stained with anti-CD11c and anti-H-2Kb-SIINFEKL antibodies for 30 min at 4°C. The percentage of H-2Kb-SIINFEKL+ cells among CD11c+ cells was determined.
[0069] The results are as follows Figure 5 The positive rates of SIINFEKL-H-2Kb in DCs treated with LNP@CARΔ and LNP@CAR were significantly higher than in other groups, with the LNP@CAR group showing the highest positive rate of SIINFEKL-H-2Kb in DCs. These findings indicate that CAR-DCs manufactured using LNP@CAR have enhanced antigen processing and presentation capabilities. In summary, these results demonstrate that CAR-DCs manufactured using LNP@CAR possess excellent uptake, processing, and presentation capabilities for tumor antigens.
[0070] In vivo anti-tumor experiment 1. Colon cancer model
[0071] This experiment used a colon cancer model as an example, designing a CAR structure targeting colon cancer scfv. CT26 cells stably expressing human CEACAM5 (CT26-hCEA-OVA) were seeded into the right axilla (1 × 10⁶ cells per mouse). 6 A mouse subcutaneous colon cancer model was established. Tumor-bearing mice were divided into groups, and on days 7 and 14 post-inoculation, they were injected via the tail vein with saline, pCAR, LNP@CAR△, or LNP@CAR. Starting on day 7 post-inoculation, tumor length (L) and short vertical diameter (W) were measured. The tumor volume was calculated using the formula: V = (L × W) 2 ) / 2, during which tumor growth was monitored in real time. Mice treated with LNP@CARΔ or LNP@CAR showed significantly reduced tumor growth rates, such as Figure 6 Among them, the LNP@CAR group showed the most significant inhibition of tumor growth.
[0072] 2. Breast Cancer Model
[0073] This experiment uses a breast cancer model as an example to design a CAR structure for targeting breast cancer scfv. For example... Figure 7As shown in Figure A, the CAR structure contains, from the 5' end to the 3' end, the following components in sequence: Zbtb46 dendritic cell-specific promoter, CD8α signal peptide, Myc tag, HER2-specific single-stranded variable fragment (HER2-scFv), CD8 hinge region, CD8 transmembrane region, CD3ζ intracellular signal transduction domain, P2A autocleavage peptide, and EGFP reporter gene.
[0074] This experiment used a breast cancer model as an example, and 4T1 cells (4T1-hHER2) stably expressing human HER2 were seeded into the right axilla (1 × 10⁶ cells per mouse). 6 A subcutaneous breast cancer model was established in mice. Mice bearing tumors were divided into groups, and on days 7 and 14 post-inoculation, they were injected via the tail vein with either saline or LNP@CAR. Starting on day 7 post-inoculation, tumor length (L) and short vertical diameter (W) were measured. The tumor volume was calculated using the formula: V = (L × W) 2 () / 2, to monitor tumor growth in real time during the experiment. For example... Figure 7 In mice with tumors in B and C, the tumor growth rate was significantly reduced by LNP@CAR treatment.
[0075] 3. Lung Cancer Model
[0076] This experiment used a lung cancer model as an example to design a CAR structure targeting the subclinical carcinoma virus (SCCV) in lung cancer. Figure 8 As shown in Figure A, the CAR structure contains, from the 5' end to the 3' end, the following components in sequence: Zbtb46 dendritic cell-specific promoter, CD8α signal peptide, Myc tag, EGFR-specific single-stranded variable fragment (EGFR-scFv), CD8 hinge region, CD8 transmembrane region, CD3ζ intracellular signal transduction domain, P2A autocleavage peptide, and EGFP reporter gene.
[0077] This experiment used a lung cancer model as an example, and injected stable LLC cells (LLC-hEGFR) expressing human EGFR (1 × 10⁶ cells per mouse) into the right axilla. 6 A subcutaneous lung cancer model was established in mice. Tumor-bearing mice were divided into groups, and on days 7 and 14 post-inoculation, they were injected via the tail vein with either saline or LNP@CAR. Starting on day 7 post-inoculation, tumor length (L) and short vertical diameter (W) were measured. The tumor volume was calculated using the formula: V = (L × W) 2 () / 2, to monitor tumor growth in real time during the experiment. For example... Figure 8 In mice with tumors in B and C, the tumor growth rate was significantly reduced by LNP@CAR treatment.
[0078] 4. Hematologic tumor model
[0079] This experiment used a hematologic malignancy model as an example to design CAR structures targeting different subcellular carcinomas (SCCFVs). For example... Figure 9 As shown in Figure A, CAR structures targeting CD19, CD20, and BCMA were constructed, respectively. Each CAR structure was driven by the Zbtb46 promoter and sequentially contained a CD8α signal peptide, a Myc tag, the corresponding target scFv, a CD8 hinge region, a CD8 transmembrane region, a CD3ζ intracellular signal transduction domain, a P2A self-cleaving peptide, and an EGFP reporter gene.
[0080] This experiment used a hematologic malignancy model as an example, and A20 cells stably expressing human CD19 (A20-hCD19-luci), A20 cells stably expressing human CD20 (A20-hCD20-luci), or A20 cells stably expressing human BCMA (A20-hBCMA-luci) were injected via tail vein (1 × 10⁻⁶ cells per mouse). 6 A mouse hematologic malignancy model was established. Mice bearing tumors were divided into groups, and on days 7 and 14 post-inoculation, they were injected via the tail vein with either saline or LNP@CAR. In vivo imaging began on day 0 post-inoculation, and mouse survival was monitored in real-time throughout the experiment. Figure 9 In mice with tumors in groups B and C, the tumor growth rate was significantly reduced and the survival rate was high after treatment with LNP@CAR.
[0081] 5. Verify the functionality of different CAR structures
[0082] To verify the impact of different CAR structural elements on functionality, this invention constructed a series of CAR structures targeting the M5A (CEA), such as... Figure 10 As shown in Figure A.
[0083] This experiment used a colon cancer model as an example, designing various CAR structures targeting colon cancer-specific scfv. CT26 cells stably expressing human CEACAM5 (CT26-hCEA-OVA) were seeded into the right axilla (1 × 10⁶ cells per mouse). 6 A subcutaneous colon cancer model was established in mice. Tumor-bearing mice were divided into groups, and on days 7 and 14 post-inoculation, they were injected via the tail vein with either saline or LNP@CAR. Starting on day 7 post-inoculation, tumor length (L) and short vertical diameter (W) were measured. The tumor volume was calculated using the formula: V = (L × W) 2 () / 2, to monitor tumor growth in real time during the experiment. For example... Figure 10 Compared with the saline group, mice treated with different CAR structures (B, C, and D) showed significantly reduced tumor growth rates and significant anti-tumor effects. Structure 3 (4-1BB+CD3ζ), containing a co-stimulatory domain, showed even better results, but no significant differences were observed among the groups, requiring further experimental verification. These results demonstrate that the CAR-DC platform of this invention has good structural adaptability.
[0084] Although the feasibility of this invention has been verified using mouse models, its design principles and technical approach can be directly applied to humans. The reasons are as follows: This invention employs a modular design, allowing for seamless replacement of gene elements used in mouse experiments with optimized human sequences; the core immunological mechanisms are highly conserved across species. The LNP delivery technology used is a mature clinical platform, and the tumor antigens targeted by this invention (such as CEA, HER2, and CD19) are entirely based on human disease settings. Therefore, the success of mouse experiments provides a solid and reliable foundation for the humanization and clinical translation of the technology, and the ultimate goal and implementation path of this invention are clearly directed towards human cancer treatment.
[0085] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A chimeric antigen receptor dendritic cell product, characterized in that, Dendritic cells containing chimeric antigen receptors (CARs), wherein the CARs are composed of the following: Antigen recognition region (scFv): The tumor antigens targeted by the scFv are CEA, HER2, EGFR, CD19, CD20, BCMA, CD22, CD30, CD33, MSLN, PSMA, GPC3, PSA, and PD-L1; Hinge region: Selected from the hinge regions of CD8, CD8α, CD28, IgG, IgG1, IgG4 or their functional variants; Transmembrane region: selected from the transmembrane regions of CD8, CD8α, CD3ζ, CD4, CD28 or their functional variants; Intracellular region: Selected from one or more of CD3ζ, FcγR, 4-1BB, CD28, OX40, ICOS, CD27 or their functional variants.
2. A chimeric antigen receptor (CAR) expression system selectively expressed in dendritic cells, characterized in that, It contains a nucleic acid molecule that can express CAR in dendritic cells (DCs). The sequence encoding CAR contains sequences encoding the following elements in sequence: single-stranded variable fragment (scFv), hinge region, transmembrane region, and intracellular signal transduction domain.
3. The expression system as described in claim 2, characterized in that, The nucleic acid molecule comprises a CAR-encoding gene in dendritic cells (DCs), wherein the 5' to 3' ends of the nucleic acid molecule comprises, in sequence, the following operatively linked genetic elements: a dendritic cell-specific promoter, a sequence encoding a chimeric antigen receptor (CAR); wherein the sequence encoding the CAR comprises, in sequence, sequences encoding the following elements: a signal peptide, a single-stranded variable fragment (scFv), a hinge region, a transmembrane region, and an intracellular signal transduction domain.
4. The expression system as described in claim 2 or 3, characterized in that, The dendritic cell-specific promoters are selected from ZBTB46 promoter, CD209 promoter, CD11c promoter, BDCA-2 (CD303) promoter or their functional variants; The signal peptide is selected from the CD8α signal peptide or its functional variants; The scFv targets tumor-associated antigens; the tumor antigens targeted by the scFv are CEA, HER2, EGFR, CD19, CD20, BCMA, CD22, CD30, CD33, MSLN, PSMA, GPC3, PSA, and PD-L1. The hinge region is selected from the hinge regions of CD8, CD8α, CD28, IgG, IgG1, IgG4 or their functional variants; The transmembrane region is selected from CD8, CD8α, CD3ζ, CD4, CD28 transmembrane regions or their functional variants; The intracellular signal transduction domain is selected from one or more combinations of CD3ζ, FcγR, 4-1BB, CD28, OX40, ICOS, CD27 or their functional variants; The nucleic acid molecule is in the form of DNA or RNA.
5. A CAR vector based on dendritic cell targeting, characterized in that, The system comprises the CAR expression system of claim 2, 3 or 4, wherein the vector is a plasmid, a lentiviral vector, an adeno-associated virus vector or an in vitro transcribed mRNA.
6. A dendritic cell-targeting lipid nanoparticle (LNP) delivery carrier, characterized in that, The system comprises the CAR expression system of claim 2, 3 or 4, which is in the form of plasmid DNA; and an LNP encapsulating the plasmid DNA.
7. A method for preparing chimeric antigen receptor dendritic cells (CAR-DCs), characterized in that, The invention includes introducing the CAR expression system of claim 2, 3, or 4, the CAR vector of claim 5, or the delivery vector of claim 6 into dendritic cells (DCs) to express the chimeric antigen receptor; the introduction is performed in vitro or in vivo; the CAR-DC is capable of initiating an immune response against the CAR-targeted antigen and against multiple tumor antigens; and / or the CAR-DC is capable of enhancing the infiltration of immune cells into tumor tissues and remodeling the tumor immunosuppressive microenvironment.
8. The use of the CAR expression system of claim 2, 3, or 4, the CAR vector of claim 5, or the delivery vector of claim 6, or the CAR-DC prepared by the method of claim 7, in the preparation of a drug for the treatment or prevention of tumors; the tumors include solid tumors or hematologic malignancies.
9. The application as described in claim 8, characterized in that, The solid tumors include colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, stomach cancer, liver cancer, glioma, and prostate cancer; the hematologic malignancies include leukemia, lymphoma, and multiple myeloma.
10. A pharmaceutical composition, characterized in that, It includes the CAR expression system of claim 2, 3 or 4, the CAR vector of claim 5, or the delivery vector of claim 6, or the CAR-DC prepared by the method of claim 7, and a pharmaceutically acceptable vector.
11. The pharmaceutical composition of claim 10, characterized in that, The pharmaceutical composition is in the form of lipid nanoparticles (LNPs), which encapsulate the CAR expression system or CAR carrier; the LNPs are prepared by the following method: (1) Preparation of lipid phase in lipid nanoparticles: Distearate phosphatidylcholine (DSPC), polyethylene glycol 2000-dimyristoylglycerol (PEG2000-DMG), ionizable cationic lipid SM-102, and cholesterol were dissolved in an organic solvent to obtain a lipid phase solution; (2) Preparation of aqueous phase in lipid nanoparticles: The CAR expression system of claim 2, 3 or 4 or the CAR vector of claim 5 is added to an acidic buffer solution to obtain an aqueous phase solution; (3) LNPs were prepared using microfluidic mixing.