A chimeric antigen receptor-modified t cell targeting fap, its preparation method and application

Abstract

The application discloses a kind of FAP targeted chimeric antigen receptor regulatory T cells, its preparation method and application, belong to biomedical technology field.The FAP targeted chimeric antigen receptor regulatory T cell contains chimeric antigen receptor, the chimeric antigen receptor includes single-chain antibody targeted to FAP, CD8 alpha hinge region, CD8 transmembrane region, CD28 signal region and CD3 zeta signal region, its nucleotide sequence is as shown in SEQ ID NO.1.The FAP targeted chimeric antigen receptor regulatory T cell can effectively accumulate in damaged heart, and control the excessive fibrosis and inflammatory response in MI, which not only highlights the therapeutic potential of CAR Tregs against FAP in promoting heart healing, but also lays the foundation for CAR Treg treatment in clinical environment of severe MI.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a chimeric antigen receptor regulatory T cell that targets FAP, its preparation method and application. Background Technology

[0002] Myocardial infarction (MI) is one of the leading causes of death worldwide, posing a significant challenge to cardiac repair. MI survivors, in particular, who miss the critical window for emergency reperfusion therapy, often face the risk of chronic heart failure (HF) due to insufficient myocardial repair. To address this urgent need, it is essential to identify and develop effective interventions that can effectively promote cardiac healing.

[0003] Regulatory T cells (Tregs) play a crucial role in immune homeostasis by suppressing innate and adaptive immune responses and have recently been found to be involved in cardiac repair after myocardial infarction (MI). Experimental studies have consistently demonstrated that increasing Treg numbers through infusion of polyclonal Tregs or the use of hyperactive anti-CD28 antibodies can improve function after cardiac injury. The key role of Tregs in regulating fibrosis and modulating inflammatory responses opens exciting possibilities for Treg-based cell therapies to enhance cardiac healing after MI. However, despite the promising results of Treg therapy, some limitations must be addressed. A major challenge is the limited number of Tregs available for treatment, as they only account for a small percentage of circulating CD4+. + T cells account for only 2-8% of the total cell count, making it difficult to obtain a sufficient number to produce a significant therapeutic effect. Furthermore, infusion of polyclonal Tregs has shown limited efficacy and carries the risk of nonspecific immunosuppression. Another obstacle is the limited availability and rarity of antigen-specific Tregs, making their generation challenging. Therefore, innovative strategies are needed to overcome these limitations in order to fully realize the potential of Tregs in the treatment of MI.

[0004] Chimeric antigen receptor (CAR) therapy combines the advantages of T-cell immunotherapy with the specificity of antibodies. Through genetic engineering, antibody-derived variable regions are combined with the signaling and co-stimulatory components of immune receptors. CARs can recognize antigens independently of MHC compatibility, eliminating the need for HLA diversity. In recent years, CARs have not only achieved significant success in treating hematologic malignancies but have also shown therapeutic potential as redirected Treg cells in disease states such as Alzheimer's disease, inflammatory bowel disease, vitiligo, multiple sclerosis, and type 1 diabetes. In preclinical studies, using mouse and humanized mouse models, CAR-Treg cell therapy has proven to be a promising cell therapy for modulating pathological immune responses and restoring immune homeostasis.

[0005] Fibroblast activating protein (FAP) is a cell surface glycoprotein primarily expressed during embryonic development and in conditions involving active tissue remodeling, such as wound healing, tissue fibrosis, and various types of tumors. Furthermore, Aghajanian et al. reported that FAP is strongly expressed in fibroblasts of ventricular tissue from patients with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), while expression is minimal in normal ventricular tissue. This study suggests that the absence of FAP in fibroblasts reduces cardiac fibrosis, highlighting the crucial role of FAP in the fibrotic process. This makes FAP an attractive candidate antigen for redirecting therapeutic immune cells to the infarcted cardiac site. Summary of the Invention

[0006] Therefore, we combined CAR technology with Tregs targeting CFs that express FAP, which may provide a more specific and safer approach to promote cardiac healing and prevent HF after MI.

[0007] One of the objectives of this invention is to provide a chimeric antigen receptor targeting FAP, wherein the chimeric antigen receptor targeting FAP includes a single-chain antibody targeting FAP, a CD8α hinge region, a CD8 transmembrane region, a CD28 signal region, and a CD3ζ signal region, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] A second objective of this invention is to provide a recombinant viral vector comprising the aforementioned chimeric antigen receptor targeting FAP.

[0009] A third objective of this invention is to provide a method for preparing the above-mentioned recombinant viral vector, the method comprising the following steps:

[0010] (1) The vector plasmid pLVX-EF1a-mCMV-ZsGreen and the fragment with the nucleotide sequence shown in SEQ ID NO.1 were ligated after double enzyme digestion to obtain the recombinant plasmid;

[0011] (2) The recombinant plasmid, packaging plasmid and envelope plasmid obtained in step (1) are co-transfected into eukaryotic cells to obtain a recombinant viral vector.

[0012] Preferably, the double enzyme digestion sites in step (1) are NotI and BamHI.

[0013] More preferably, the eukaryotic cells in step (2) are HEK293T cells.

[0014] A fourth objective of this invention is to provide a host cell comprising the aforementioned recombinant viral vector.

[0015] The fifth objective of this invention is to provide a chimeric antigen receptor regulatory T cell that targets FAP, wherein the regulatory T cell contains the aforementioned chimeric antigen receptor that targets FAP.

[0016] The sixth objective of this invention is to provide a method for preparing chimeric antigen receptor regulatory T cells targeting FAP, the method comprising the following steps: transducing Treg cells with a recombinant viral vector obtained by the above method.

[0017] The seventh objective of this invention is to provide the application of the above-mentioned chimeric antigen receptor targeting FAP, or the above-mentioned recombinant viral vector, or the above-mentioned host cell, or the above-mentioned chimeric antigen receptor regulatory T cell targeting FAP in the preparation of a cardiac treatment product.

[0018] Preferably, the product is a drug.

[0019] The inventors used a CAR construct targeting FAP to redirect Tregs to infarcted cardiac tissue. We found that these FAP CAR Tregs effectively accumulated in the damaged heart and controlled excessive fibrosis and inflammatory responses in MI. This is the first groundbreaking study to not only highlight the therapeutic potential of FAP-targeted CAR Tregs in promoting cardiac healing but also to lay the foundation for CAR Treg therapy in severe MI in the clinical setting.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] CAR Treg technology, through its specific targeting of the fibroblast-activating protein FAP, directly acts on key cells in the cardiac repair process, overcoming the limitations of widespread immunosuppression in traditional cardiac treatments and improving the precision and safety of treatment. CAR Tregs reduce cardiac fibrosis, maintain cardiac structure and function, thereby mitigating the degree of cardiac remodeling. Simultaneously, CAR Tregs help reduce cardiac inflammation, promote cardiac tissue repair, and improve the quality of cardiac healing by releasing anti-inflammatory and repairing cytokines such as IL10 and TGFβ. Furthermore, the specific action of CAR Tregs reduces the potentially widespread side effects of traditional immunosuppressants, as they target only specific pathological processes rather than the broader immune system. When administered via tail vein injection during the critical period after myocardial infarction, CAR Tregs can form significant cell clusters in the damaged cardiac region, directly participating in cardiac repair, improving treatment efficacy, and reducing potential impacts on other organs. The preparation and application of CAR Tregs hold promise for clinical implementation, not only reducing treatment complexity but also improving its feasibility in different medical settings. In summary, CAR Treg technology has shown significant beneficial effects in the treatment of heart disease. These effects are achieved through innovative improvements in various aspects, including specific targeting, regulation of inflammation and fibrosis, promotion of cardiac healing, reduction of toxic side effects, and enhancement of efficacy. Attached Figure Description

[0022] Figure 1 The image shows the spectrum of the vector plasmid pLVX-EF1a-mCMV-ZsGreen in Example 1.

[0023] Figure 2 The transfection efficiency (green fluorescent protein GFP) of FAP CAR Treg (FCTR) and Mock Treg (MKTR) was detected by flow cytometry in Example 1. + (Cell percentage).

[0024] Figure 3 In Example 2, during week 2 of MI, ultrasound was used to detect left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-diastolic volume (LVEDV) in the experimental and control groups. The ratio of heart weight to body weight (HW / BW) and the ratio of heart weight to tibia length (HW / TL) were also calculated.

[0025] Figure 4In Example 2, wheat germ lectin (WGA) and cardiac troponin I (cTnI) immunofluorescence staining were used to detect the cross-sectional area of ​​myocardial cells in the myocardial infarction junction of each group at week 2 of MI, and real-time quantitative PCR was used to detect the expression of myocardial hypertrophy-related genes: B-type natriuretic peptide (BNP), A-type natriuretic peptide (ANP), and β-myosin heavy chain (βMHC).

[0026] Figure 5 In Example 2, Masson staining was used to detect fibrosis in the infarct junction at week 2 of myocardial infarction, and real-time quantitative PCR was used to detect the expression of fibrosis-related genes: α-smooth muscle actin (αSMA) and type I collagen α1 chain (col1a1).

[0027] Figure 6 In Example 2, real-time quantitative PCR was used to detect the expression of inflammation-related genes: interleukin-1β (IL1β), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα), and flow cytometry was used to detect the number of effector T cells and macrophages infiltrating the heart.

[0028] Figure 7 In Example 2, immunofluorescence staining of α-smooth muscle actin (αSMA) and vimentin was used to detect the proportion of myofibroblasts in the myocardial infarction junction area (αSMA+Vim+ / Vim+%) and the total proportion of fibroblasts (Vimentin+%) in each group at week 1 of MI.

[0029] Figure 8 The proportion of different types of collagen was detected under polarized light using Sirius red staining in Example 2.

[0030] Figure 9 The results of α-smooth muscle actin (αSMA), Ki67 and TUNEL fluorescence staining in Example 2 were used to detect the proliferation or apoptosis of myofibroblasts. Detailed Implementation

[0031] Example 1: Application of FAP CAR Treg cells in the treatment of cell damage

[0032] The application includes CAR Treg cells and FAP bound to them. On the one hand, the cells target the damaged area through CAR Treg cells to regulate local inflammation in the heart injury area. On the other hand, they inhibit the transdifferentiation of FAP-expressing fibroblasts into myofibroblasts, thereby inhibiting excessive fibrosis of the heart.

[0033] The preparation and detection of chimeric antigen receptor regulatory T cells targeting FAP provided in this embodiment includes the following steps:

[0034] (1) Molecular structure design

[0035] Construct the FAP-CAR gene sequence (SEQ ID NO.1) targeting FAP, including a single-chain antibody targeting FAP, the CD8α hinge region, the CD8 transmembrane region, the CD28 signaling region, and the CD3ζ signaling region:

[0036] SEQ ID NO.1:

[0037]

[0038] (2) Constructing the pLVX-EF1a-FAP-CAR-mCMV-ZsGreen plasmid

[0039] ①DNA primer design and synthesis:

[0040] FAP-CAR-NotI-F (SEQ ID NO.2):

[0041] 5'-ATTTGCGGCCGCCACCATGGCCTTACCAGTGACCGCCTTGC-3';

[0042] FAP-CAR-BamHI-R (SEQ ID NO.3):

[0043] 5'-CGCGGATCCTCAGCGAGGGGGCAGGGCCTGCATGTGA-3';

[0044] ② Using a plasmid containing FAP-CAR DNA as a template, the target gene FAP-CAR was amplified by PCR. NotI and BamHI restriction sites were introduced at both ends of the primers, respectively. The target gene FAP-CAR DNA band (SEQ ID NO.1, 1479bp) was recovered by 1% agarose gel.

[0045] ③ Vector plasmid pLVX-EF1a-mCMV-ZsGreen (see plasmid map) Figure 1 (This plasmid was provided by Shanghai Dianjun Biotechnology Co., Ltd.) NotI and BamHI Double enzyme digestion

[0046] The enzyme digestion reaction was carried out in a water bath at 37°C for 3 hours. The vector enzyme digestion system is as follows:

[0047] Table 1

[0048]

[0049] The large fragment of plasmid pLVX-EF1a-mCMV-Zs was recovered by 1% agarose gel electrophoresis after digestion with Green NotI+BamHI.

[0050] ④ Target gene FAP-CAR NotI and BamHI Double enzyme digestion

[0051] The enzyme digestion reaction was carried out in a water bath at 37°C for 3 hours. The enzyme digestion system is as follows:

[0052] Table 2

[0053]

[0054] The FAP-CAR NotI+BamHI digested fragments were recovered by 1% agarose gel electrophoresis.

[0055] ⑤ The large fragment recovered from plasmid pLVX-EF1a-mCMV-ZsGreen was ligated to the FAP-CAR fragment. The ligation reaction was carried out at 22℃ for 3 hours. The ligation reaction system is as follows:

[0056] Table 3

[0057]

[0058] ⑥ Transformation of ligation product: Mix 10 μL of ligation product with 100 μL of JM109 competent bacteria, incubate on ice for 30 min, heat shock at 42°C for 45 s, immediately place on ice for 2 min, add 400 μL of LB medium preheated to room temperature, incubate at 37°C in a shaker for 1 h, centrifuge at 4000 rpm for 1 min, discard 400 μL of culture supernatant, mix the remaining 100 μL with a pipette and spread evenly on LB plates containing 100 μg / ml Ampicillin resistance, and incubate in an inverted incubator at 37°C overnight.

[0059] ⑦ Pick 3 single colonies and inoculate them into LB medium containing 5 ml of 100 μg / ml Ampicillin resistance. Incubate overnight in a shaker at 250 rpm and 37°C. Extract plasmids using a small-volume plasmid extraction kit. Perform enzyme digestion identification using SpeI+BamHI. Select positive clones that are correctly identified by enzyme digestion for sequencing verification.

[0060] (2) Lentiviral preparation and lentivirus packaging

[0061] The recombinant plasmid pLVX-EF1a-FAP-CAR-mCMV-ZsGreen, the packaging plasmid psPAX2, and the envelope plasmid p-EcoEnv were co-transfected into cultured HEK293T cells. The medium was replaced with complete medium without P / S the day before transfection. Then, the lentiviral vector plasmid and packaging plasmid were co-transfected into the cells using a CPT high-efficiency transfection kit. After transfection, the cells were cultured for 24 hours, and the medium was replaced with fresh medium. After 48 hours, the supernatant containing the virus was collected, and cell debris and impurities were removed by centrifugation and filtration. Finally, the virus particles were resuspended by ultracentrifugation and in an appropriate medium, aliquoted, and stored at -80°C for later use.

[0062] The biological titer of lentiviruses was determined by infecting HEK 293 cells and counting fluorescent cells. Before the experiment, HEK 293 cells were seeded into 48-well plates, and the virus was serially diluted using complete medium containing polybrene. The diluted virus solutions were then added to the cell wells. After overnight infection, the medium was replaced with fresh medium, and the cells were cultured for another 48 hours. The number of fluorescently positive cells was observed and counted using a fluorescence microscope. Based on the number of fluorescent cells and the dilution factor, the viral titer, i.e., the number of transduction units per microliter of viral solution (TU / ml), was calculated.

[0063] The control group, Mock Treg, contained only ZsGreen fluorescence and lacked the FAP CAR structure. The plasmid pLVX-EF1a-mCMV-ZsGreen, the packaging plasmid psPAX2, and the envelope plasmid p-EcoEnv were co-transfected into cultured HEK293T cells.

[0064] (3) Separate CD4 + CD25 + Treg cells

[0065] Mouse spleen Treg cells were isolated using Miltenyi magnetic beads, and the purity of the Tregs was assessed by flow cytometry, with a purity exceeding 90%.

[0066] (4) Activate Treg cells

[0067] Tregs were cultured in 2 ml of T cell culture medium at a density of 2 × 10^6 cells per well in 12-well plates. The Tregs were cultured together with 1000 U / mL of recombinant IL-2 and T cell activator anti-CD3 / CD28 magnetic beads (ratio 2:1).

[0068] (5) Lentiviral transduction of Treg cells to prepare FAP CAR Treg cells

[0069] After culturing Tregs for 48 hours, the cells were transferred to 24-well plates pre-coated with retrolectin (50 μg / ml) and centrifuged at 1000G for 1 hour. Subsequently, the cells were expanded for 72–96 hours in a solution of 50 U / mL IL-2.

[0070] (6) After preparation, FACS was used to detect the transduction efficiency (GFP positivity rate) of mouse FAP CAR Treg cells, such as... Figure 2As shown, the transfection efficiency was 60%-65%. Flow cytometry was used to compare the expression of CD4, CD25, FOXP3, and CTLA-4 in FAP CAR Tregs, Mock Tregs, and uninfected Tregs, revealing that lentiviral transduction did not alter the inhibitory phenotype of the Tregs themselves.

[0071] In addition, to verify the effect of FAP-specific activation of FAP CAR Tregs, co-culture experiments were conducted in four groups: (1) co-culture of Mock Tregs with 3T3 cell lines; (2) co-culture of Mock Tregs with 3T3.FAP cells; (3) co-culture of FAP CAR Tregs with 3T3 cell lines; and (4) co-culture of FAP CAR Tregs with 3T3.FAP cells. The 3T3.FAP cells were obtained by transfecting the 3T3 cell line with a recombinant plasmid overexpressing mouse FAP and tagged with a flag using lipo3000. After 24 hours, the results showed that FAP CAR Tregs effectively activated 3T3.FAP fibroblasts, exhibiting increased expression of CD69, GARP, and LAP, a response not observed in Mock Tregs. These findings highlight the FAP-specific activation of FAP CAR Tregs.

[0072] Example 2

[0073] In this embodiment, male c57 mice aged 8-12 weeks were used to establish a myocardial infarction (MI) model. The MI model was established by permanently ligating the left anterior descending coronary artery. In short, this process involved anesthetizing the mice with ketamine (50 mg / kg) and sodium pentobarbital (50 mg / kg) via intraperitoneal injection, followed by endotracheal intubation and connection to a rodent ventilator to ensure physiological stability during the procedure. The heart was accessed via a left thoracotomy, and the left anterior descending coronary artery was carefully ligated using fine 6-0 sutures. On the third day after model establishment, CAR Treg cells (2.5 × 10^5 cells per mouse) were injected via the tail vein.

[0074] This embodiment sets up an experimental group and a control group. The experimental group uses FAP CAR Treg prepared in Example 1; the control group uses Mock Treg cells transduced with empty lentivirus and PBS solution for the experiment.

[0075] Two weeks after MI modeling, small animal ultrasound (Vevo3100) was used to image the short axis of the ventricles and the long axis of the sternum in both experimental and control mice. We measured the short axis M-mode images of the heart captured at the mid-papillary muscle level, including left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-diastolic volume (LVEDV).

[0076] like Figure 3 As shown, compared with the Mock Treg cells and PBS solution group, FAP CAR Treg cells significantly improved cardiac function after myocardial infarction. This was manifested in increased EF and FS, and decreased LVEDV and LVEDD. In addition to the improvement in cardiac function, we also observed that MI mice treated with FAP CAR Tregs had improved heart weight to body weight ratio and heart weight to tibia length ratio compared with mice treated with the control solvent, while mice treated with Mock Tregs did not (see [link to data]). Figure 3 ).

[0077] Immunostaining with cardiac troponin I and malt germ agglutinin (WGA) showed that on day 14 after MI modeling, the cross-sectional area of ​​cardiomyocytes was smaller in the group treated with FAP CAR Tregs compared to the PBS solution group, while there was no significant reduction in the cross-sectional area of ​​Mock Tregs treated with FAP CAR Tregs. This was accompanied by a simultaneous decrease in the expression of mast genes such as BNP, ANP, and βMHC (see [link to relevant documentation]). Figure 4 ).

[0078] Masson trichrome staining of the periinfarct area showed a significant reduction in cardiac fibrosis compared to mice treated with Mock Tregs or those treated with PBS solvent, accompanied by decreased expression of αSMA and Col1a1 genes. Figure 5 ).

[0079] Furthermore, we observed reduced expression of inflammatory factors (IL1β, IL6, and TNFα) in damaged hearts treated with FAP CAR Tregs, and a decrease in the number of effector T cells in the infarcted myocardium 7 days after MI. Simultaneously, FAP CAR Treg injection also reduced the number of infiltrating macrophages. Figure 6 Therefore, our findings consistently demonstrate that CARTregs targeting FAP have significant anti-inflammatory effects.

[0080] On day 7 after myocardial infarction (MI), immunofluorescence staining showed a significant reduction in the number of myofibroblasts expressing αSMA and Vimentin in the FAP CAR Treg treatment group. The total number of CFs expressing Vimentin was consistent across the three groups. Figure 7Furthermore, Sirius red polarized light microscopy analysis showed that MI mice treated with FAP CAR Tregs exhibited a reduction in tightly packed, rigid collagen fibers (orange-red) within the infarct scar. Figure 8 ).

[0081] To determine whether the decrease in myofibroblast number in FAP CAR Tregs-treated scar tissue was due to reduced proliferation or increased apoptosis, we performed immunostaining with proliferation (Ki67) and apoptosis (TUNEL) markers. We observed αSMA cells expressing either Ki67 or TUNEL. + Vim + The proportion of CFs did not differ significantly. Figure 9 These findings suggest that the reduction in the number of myofibroblasts and the reduction in cardiac fibrosis in scar tissue treated with FAP CAR Tregs, compared to Mock Tregs and control solvent-treated MI mice, may be due to reduced myofibroblast differentiation or formation, rather than proliferation or apoptosis.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A chimeric antigen receptor targeting FAP, characterized in that, The FAP-targeted chimeric antigen receptor comprises a single-chain antibody targeting FAP, a CD8 alpha hinge region, a CD8 transmembrane region, a CD28 signal region, and a CD3 zeta signal region, and a nucleotide sequence encoding the chimeric antigen receptor is shown as SEQ ID NO.

1.

2. A recombinant viral vector, characterized in that, The recombinant viral vector comprises the FAP-targeted chimeric antigen receptor as described in claim 1.

3. A method of producing the recombinant viral vector of claim 2, wherein, The method comprises the following steps: (1) a recombinant plasmid is obtained by ligating a vector plasmid pLVX-EF1a-mCMV-ZsGreen and a fragment with a nucleotide sequence shown as SEQ ID NO. 1 after double enzyme digestion; (2) a recombinant viral vector is obtained by co-transfecting eukaryotic cells with the recombinant plasmid obtained in step (1), a packaging plasmid, and an envelope plasmid.

4. The method of claim 3, wherein, In step (1), the double enzyme digestion sites are NotI and BamHI.

5. The method of claim 4, wherein, In step (2), the eukaryotic cells are HEK293T cells.

6. A host cell, characterized in that, The host cell comprises the recombinant viral vector as described in claim 2.

7. A chimeric antigen receptor-modified T cell targeting FAP, characterized in that, The regulatory T cell contains the FAP-targeted chimeric antigen receptor as described in claim 1.

8. A method of manufacturing a chimeric antigen receptor-modified T cell targeting FAP, characterized in that, The preparation method comprises the following step: transducing Treg cells with the recombinant viral vector prepared by the method of any one of claims 3-5.

9. Use of the FAP-targeted chimeric antigen receptor of claim 1, the recombinant viral vector of claim 2, the host cell of claim 6, or the FAP-targeted chimeric antigen receptor regulatory T cell of claim 7 in the preparation of a myocardial infarction treatment product.

10. Use according to claim 9, characterized in that, The product is a drug.

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