Method for enhancing mechanical force of CAR-T cells on target cells to overcome mechanical immune checkpoints of SFs and application

Abstract

The invention discloses a method for enhancing mechanical force of CAR-T cells on target cells to overcome mechanical immune checkpoints of SFs and application, and relates to the technical field of crossing of immunotherapy and biomechanics, in particular to CAR-T cells targeting fibroblast activated protein. The invention discloses a CAR-T (chimeric antigen receptor T-T) cell and SFs (small-form-factor s) DNA (deoxyribonucleic acid) nano connector for connecting the CAR-T cell with the SFs, the formation of a CAR cluster and mechanical force transmission are promoted by shortening the membrane spacing of an immune synaptic interface to 16-28nm, the DNA nano connector is formed by self-assembling complementary tetrahedral DNA structures, and the TDS comprises a single-stranded DNA vertex S4 with the length of 24-58 nucleotides. According to the method for enhancing the mechanical force of the CAR-T cells on the target cells to overcome the mechanical immune checkpoints of the SFs and the application, the escape and killing problems of the soft target cells are solved, the soft target cells are efficiently removed, and the killing rate of the CAR-T cells treated by DNJ-17 on the SFs is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy and biomechanics, specifically to a method and application for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanical immune checkpoints of SFs. Background Technology

[0002] Myocardial fibrosis is a key pathological feature of heart disease, caused by the abnormal deposition of extracellular matrix by activated cardiac fibroblasts, ultimately accelerating the progression of heart failure. Although CAR-T cells targeting fibroblast activating protein (FAP) can specifically eliminate activated cardiac fibroblasts, clinical studies have shown that some soft-activated cardiac fibroblasts (SFs) can evade the killing effect of anti-FAP CAR-T cells by impairing the mechanical properties of CAR-T cells, subsequently leading to recurrence of myocardial fibrosis.

[0003] Traditional CAR-T cell therapy mainly relies on biochemical signal activation, while neglecting the role of mechanomechanical forces in immune synapses (IS). Studies have shown that the mechanomechanical transmission between CAR-T cells and target cells is a key factor in killing efficiency. However, SFs can evade killing by reducing membrane tension and inhibiting the pore-forming ability of perforin. Therefore, developing a strategy that can enhance the mechanomechanical force of CAR-T cells on target cells to overcome the soft mechanomechanical immune checkpoints of target cells has important clinical significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanical immune checkpoints of SFs, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method and application for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs, including CAR-T cells targeting fibroblast activation proteins; and DNA nanolinkers connecting CAR-T cells and SFs, which promote the formation of CAR clusters and the transmission of mechanical force by shortening the intermembranes of the immune synaptic interface to 16-28 nm.

[0006] Furthermore, the DNA nanolinker is self-assembled from complementary tetrahedral DNA structures, and the TDS includes: a single-stranded DNA vertex S4 with a length of 24-58 nucleotides; and three bottom NHS ester modification sites for covalently anchoring the cell membrane.

[0007] Furthermore, the TDS includes three specifications: TDS-28, TDS-23, and TDS-17, with corresponding theoretical film spacings of 28.2 nm, 22.7 nm, and 16.6 nm, respectively.

[0008] Furthermore, the CAR-T cells are anti-FAP CAR-T cells containing 4-1BB or CD28 co-stimulatory domains.

[0009] Furthermore, one end of the DNA nanolinker is anchored to the CAR-T cell membrane via NHS ester, and the other end is modified with an anti-FAP antibody to specifically bind to target cells.

[0010] Furthermore, its preparation method includes the following steps: (1) Synthesize a tetrahedral DNA structure with a single-stranded vertex; (2) Complementary TDS were modified onto the membranes of CAR-T cells and target cells, respectively; (3) DNA nanolinkers are formed through DNA hybridization, shortening the intercellular membrane distance to 16-28 nm.

[0011] Furthermore, in step (2), PLL-g-Dex is used to promote the membrane anchoring stability of TDS in serum-containing medium.

[0012] Furthermore, in step (3), TDS-17 is preferably used to construct DNJ-17 to achieve a membrane spacing of 16.6 nm. Further, an application of a method to enhance the mechanical force of CAR-T cells on target cells to overcome SFs mechanoimmune checkpoints, using the aforementioned method to enhance the mechanical force of CAR-T cells on target cells to overcome SFs mechanoimmune checkpoints, the treatment method can: enhance the mechanical force exerted by CAR-T cells on soft-activated cardiac fibroblasts at immune protrusions; promote perforin pore formation and granzyme B delivery; prevent myocardial fibrosis recurrence, with the therapeutic effect lasting at least 12 months.

[0013] Furthermore, the target cells are activated cardiac fibroblasts with a stiffness of less than 164 Pa.

[0014] Compared with existing technologies, this invention has the following beneficial effects: This invention solves the problem of soft target cells escaping killing, efficiently eliminates soft target cells, and significantly improves the killing rate of DNJ-17-treated CAR-T cells against SFs, eliminating their competitive survival advantage. It is the first to incorporate mechanical force regulation into CAR-T cell therapy design. It achieves precise control of membrane spacing through DNA nanotechnology, enhancing CAR aggregation and downstream mechanical signals. It also inhibits fibrosis recurrence in the long term. In a mouse model of hypertensive myocardial fibrosis, myocardial fibrosis did not recur after 12 months of treatment with CAR-T cells enhanced by CAR aggregation. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the establishment of the cardiac fibroblast model of the present invention and a partial time-point analysis data graph; Figure 2 This image shows the in vitro co-culture of anti-FAP CAR-T cells and FAP+ fibroblasts of this invention, along with some of the corresponding results. Figure 3 This is a graph showing the results of the analysis of CAR-T cell mechanical forces and the verification of the relationship between membrane tension and perforin function in this invention. Figure 4 The figure shows the experimental results verifying whether increasing CAR-T cell activity leads to increased cytotoxicity in this invention. Figure 5 The basic structural diagram of the DNA nanolinkers (DNJs) of this invention is shown below; Figure 6 This is an experimental diagram illustrating the modification and biocompatibility of the cell membrane during the cell membrane anchoring of the DNJ of this invention. Figure 7 This diagram illustrates the measurement and functional verification of the DNJ-controlled membrane spacing according to the present invention. Figure 8 This is an experimental diagram illustrating the mechanism by which DNJ enhances CAR-T cell function in this invention. Figure 9 This is an engineering modification diagram of DNJ-17 for in vivo application of the DNA nanolinker (DNJ) of the present invention. Detailed Implementation

[0016] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0017] like Figures 1 to 9 As shown, a method and its application for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanical immune checkpoints of soft fibroblasts are described. The specific operation is as follows: Verification of the escape mechanism of soft fibroblasts: 1. In vivo model validation 1) See Figure 1 a. Establishment of a myocardial fibrosis model: The experimental group was induced with myocardial fibrosis in mice by continuous infusion of angiotensin II (Ang II) and phenylephrine (A / P) via an osmotic pump (1 week). The control group was infused with saline.

[0018] 2) See Figure 1 b. CAR-T cell therapy: Week 1, first infusion of anti-FAP CAR-T cells (containing 4-1BB co-stimulatory domain); Week 2, second infusion to enhance efficacy; control group received PBS or null CAR-T cells.

[0019] 3) See Figure 1c. Time point analysis: Short-term (week 4) assessment of fibrosis degree (Masson staining), cardiac function (echocardiography), and FAP+ fibroblast ratio (flow cytometry); long-term (week 24) observation of fibrosis recurrence and dynamic changes in FAP+ fibroblasts.

[0020] 2. In vitro co-culture experiment (see...) Figure 2 a) 1) See Figure 2 b. Cell isolation and culture: Activated FAP+ fibroblasts (BFs) were isolated from A / P-treated mouse hearts. A PEG hydrogel coated with decellularized dECM matrix of the damaged heart was used to mimic the in vivo mechanical microenvironment of activated cardiac fibroblasts.

[0021] 2) See Figure 2 c. CAR-T cell co-culture: BFs are co-cultured with anti-FAP CAR-T cells at different ratios (e.g., 1:1, 1:5) or for different times (24 / 48 hours), and the surviving activated cardiac fibroblasts are defined as SFs.

[0022] 3) Killing efficiency detection: We used flow cytometry to quantify the survival rate of BFs and SFs (Annexin V / PI staining), and then compared the differences in killing effect of CAR-T cells on BFs and SFs.

[0023] 3. Verification of Escape Mechanisms 1) Establish the core hypothesis: SFs reduce the mechanical force on CAR-T cells by decreasing stiffness, thereby inhibiting the formation of perforin channels.

[0024] 2) Cell stiffness detection: Atomic force microscopy (AFM) was used to measure the elastic modulus of BFs and SFs.

[0025] 3) See Figure 3 a. Mechanical force analysis of CAR-T cells: The traction force of CAR-T cells was quantified by traction force microscopy (TFM) on hydrogels of different stiffness (164-557 Pa); and in the detection of Pyk2 phosphorylation, the pPyk2 level of CAR-T cells co-cultured with BFs / SFs was compared by Western blot.

[0026] 4) See Figure 3 b. Verification of the relationship between membrane tension and perforin function: In the membrane tension regulation experiment, SFs were placed in hypotonic (increase tension) or hypertonic (decrease tension) medium, and perforin and granzyme B (GrzmB) were added and incubated together. The influx of GrzmB was detected by fluorescently labeled antibody.

[0027] 4. Validation of intervention strategies: 1) Target cell sclerosis: SFs were treated with the actin polymerizer Jasplakinolide (Jas), and the stiffness change was detected by AFM and the killing efficiency of CAR-T cells was detected by LDH release assay.

[0028] 2) See Figure 4 To enhance the mechanical force of CAR-T cells: CAR-T cells were pretreated with actin polymerization inhibitors (cytochalasin D, CytoD) and myosin inhibitors (Blebbistatin, Bleb) to inhibit cellular force generation. TFM was used to assess traction force and killing efficiency.

[0029] 3) CAR aggregation enhancement strategy: Promote the formation of CAR aggregation after antigen binding through engineered CAR design, and then use confocal microscopy to observe the correlation between the size of the formed CAR aggregation and the matrix stiffness.

[0030] Design and application of DNA nanolinkers (DNJs): 1. Design of DNA Nanolinkers (DNJs) 1) See Figure 5 a. Basic structure: DNJ is formed by DNA hybridization of two complementary tetrahedral DNAs (TDS). The TDS structure consists of a bottom apex modified with three NHS esters (for covalently coupling amino groups on the cell membrane) and a top apex with a single-stranded overhang (ssDNA overhang) for hybridization with the complementary TDS. There are three types of DNJ: DNJ-28 ​​(theoretical height 28.2 nm) formed by hybridization of two TDS-28s, DNJ-23 (theoretical height 22.7 nm) formed by hybridization of two TDS-23s, and DNJ-17 (theoretical height 16.6 nm) formed by hybridization of two TDS-17s.

[0031] 2) See Figure 5 b. Synthesis and verification: First, TDS is assembled by adding DNA strands step by step and verifying the assembly efficiency using gel electrophoresis (PAGE). Then, the three-dimensional structure of TDS is confirmed using atomic force microscopy (AFM). Second, complementary TDS (such as TDS-28 and complementary TDS-28) are mixed and the hybridization efficiency is verified by fluorescent labeling (such as FAM / Cy3).

[0032] 2. Cell membrane anchoring of DNJ 1) See Figure 6a. Cell membrane modification: NHS esters were covalently bound to amino groups on the cell membrane surface (it needs to be verified that NHS-free TDS does not bind). PLL-g-Dex (polylysine-dextran copolymer) was then used to enhance the stability of TDS in serum-containing culture medium. Subsequently, the distribution of FAM-labeled TDS on the CAR-T cell membrane was observed using a fluorescence microscope, and the fluorescence intensity of membrane-bound TDS was quantified by flow cytometry to verify the anchoring efficiency.

[0033] 2) See Figure 6 b. Biocompatibility verification: The effects of TDS modification on cell viability (CCK-8), surface protein expression (flow cytometry), and CAR-T cell function (IFN-γ secretion) were examined.

[0034] 3. Experimental verification of DNJ modulation of membrane spacing 1) See Figure 7 a. Membrane spacing measurement: TDS-modified CAR-T cells were co-incubated with FAP-modified SLB (soft / hard hydrogel) (SLB coated with complementary TDS); membrane spacing was quantified using SAIM: DNJ-28: ~26.5 nm, DNJ-23: ~21.9 nm, DNJ-17: ~17.4 nm.

[0035] 2) See Figure 7 b. Functional validation: The CAR-FAP binding rate was detected using a DNA tension sensor (hairpin structure containing Cy3 fluorophore) (fluorescence dequenching); the size of CAR aggregation was observed using confocal microscopy (anti-CAR antibody labeling).

[0036] 4. Mechanism by which DNJ enhances CAR-T cell function 1) See Figure 8 a. Signaling pathway activation: First, immunofluorescence was used to detect the aggregation of ZAP70 in the immune synapse (IS) of the DNJ-17 group, then Western blot was used to detect pLAT level, and finally phalloidin staining was used to compare F-actin density.

[0037] 2) See Figure 8 b. The mechanical traction force of CAR-T cells on a soft hydrogel matrix (elastic modulus 164 Pa) was detected by the fluorescent microsphere displacement method, and the expression level of phosphorylated Pyk2 (pPyk2) was quantitatively analyzed by Western blot.

[0038] 3) See Figure 8c. Enhanced killing efficiency: The influx of granzyme B (GrzmB) was detected using fluorescently labeled antibodies to reflect the function of perforin; the apoptosis rate of SFs was quantified by LDH release or Annexin V / PI staining, and a competitive experiment was set up to eliminate the survival advantage of SFs under CAR-T pressure using DNJ-17.

[0039] 5. In vivo application design 1) See Figure 9 The engineered modification of DNJ-17 involved coupling one end to the CAR-T cell membrane (NHS ester) and the other end to an anti-FAP antibody, directly targeting SFs (without requiring SLB). This was used to verify the in vivo membrane spacing shortening effect.

[0040] 2) Animal model validation: In the myocardial fibrosis model, after A / P induced fibrosis, DNJ-17 modified CAR-T cells were infused, and then Masson staining, ultrasound, and flow cytometry were used to evaluate the efficacy of fibrosis area, cardiac function, and residual SFs.

[0041] In summary, this invention, on the one hand, utilizes DNA nanolinkers to shorten the intermembranous distance at the immune synapse (IS) interface, significantly enhancing the mechanical force exerted by CAR-T cells on target cells and the efficiency of perforin channel formation, thereby efficiently clearing soft-activated cardiac fibroblasts (SFs). On the other hand, it demonstrates the ability to achieve long-term remission and relapse-free treatment of myocardial fibrosis. Combining these two aspects provides a novel mechanical regulatory dimension for CAR-T cell therapy research, thus offering technical support for a deeper understanding of the mechanisms of disease occurrence and development, as well as regenerative medicine and biomechanical theories.

[0042] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs, characterized in that, These include CAR-T cells that target fibroblast activation proteins; and DNA nanolinkers that connect CAR-T cells to SFs, which promote CAR cluster formation and mechanotransmission by shortening the intermembranous distance at the immune synaptic interface to 16-28 nm.

2. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 1, characterized in that, The DNA nanolinker is self-assembled from complementary tetrahedral DNA structures. The TDS includes: a single-stranded DNA vertex S4 with a length of 24-58 nucleotides; and three bottom NHS ester modification sites for covalently anchoring the cell membrane.

3. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 2, characterized in that, The TDS includes three specifications: TDS-28, TDS-23, and TDS-17, with corresponding theoretical film spacings of 28.2 nm, 22.7 nm, and 16.6 nm, respectively.

4. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 3, characterized in that, The CAR-T cells are anti-FAP CAR-T cells containing 4-1BB or CD28 co-stimulatory domains.

5. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 4, characterized in that, One end of the DNA nanolinker is anchored to the CAR-T cell membrane via NHS ester, and the other end is modified with an anti-FAP antibody to specifically bind to target cells.

6. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 5, characterized in that, Its preparation method includes the following steps: (1) Synthesize a tetrahedral DNA structure with a single-stranded vertex; (2) Complementary TDS were modified onto the membranes of CAR-T cells and target cells, respectively; (3) DNA nanolinkers are formed through DNA hybridization, shortening the intercellular membrane distance to 16-28 nm.

7. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 6, characterized in that, In step (2), PLL-g-Dex is used to promote the membrane anchoring stability of TDS in serum-containing medium.

8. The method for enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs according to claim 6, characterized in that, In step (3), TDS-17 is preferably used to construct DNJ-17 to achieve a film spacing of 16.6 nm.

9. An application of enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs, comprising the method of enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs as described in any one of claims 1-8, characterized in that, The treatment method can: enhance the mechanical force exerted by CAR-T cells on soft-activated cardiac fibroblasts at immune prominences; promote perforin pore formation and granzyme B delivery; It prevents the recurrence of myocardial fibrosis, and the therapeutic effect lasts for at least 12 months.

10. The application of claim 9 in enhancing the mechanical force of CAR-T cells on target cells to overcome the mechanoimmune checkpoints of SFs, characterized in that, The target cells are activated cardiac fibroblasts with a stiffness of less than 164 Pa.

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