CN118702824A - A chimeric antigen receptor targeting discoidin domain receptor 2, a chimeric antigen receptor cell, and a preparation method and application thereof - Google Patents

Abstract

The present invention relates to a chimeric antigen receptor targeting discoidin domain receptor 2, a chimeric antigen receptor cell, and a preparation method and application thereof, wherein the chimeric antigen receptor comprises an antigen recognition domain, a connection region and an intracellular domain, and the antigen recognition domain comprises a nanobody that recognizes discoidin domain receptor 2. The present invention enhances the ability of macrophages to treat fibrotic diseases, especially lung-related fibrotic diseases, and lung transplant-related rejection and other diseases by preparing chimeric antigen receptor cells targeting discoidin domain receptor 2, preferably chimeric antigen receptor macrophages targeting discoidin domain receptor 2, and has high safety and broad application prospects.

Description

A chimeric antigen receptor targeting discoidin domain receptor 2, a chimeric antigen receptor cell, and a preparation method and application thereof

Technical Field

The present invention relates to the field of biomedicine, and in particular to a chimeric antigen receptor targeting discoidin domain receptor 2, a chimeric antigen receptor cell, and a preparation method and application thereof.

Background Art

Chimeric Antigen Receptor (CAR) technology has made significant breakthroughs in the field of cancer treatment in recent years, especially in the treatment of hematological tumors. Macrophages, as key phagocytes in the body, play the role of clearing a variety of pathogens such as bacteria, viruses and tumor cells. They not only have excellent phagocytic and digestive abilities, but also have the ability to present antigens, which can stimulate the response of the adaptive immune system and play a vital role in biological processes such as tissue repair, inflammation control and immune regulation. As a core member of the innate immune system, macrophages have gradually become an emerging focus in CAR therapy research due to their versatility and key role in the tumor microenvironment. Preliminary research results show that CAR-macrophage (CAR-M) therapy has shown significant potential in the treatment of hematological and non-hematological tumors. At present, two CAR-macrophage therapies, CT-0508 and MCY-M11, have been approved by the U.S. Food and Drug Administration (FDA) and have officially entered the clinical trial stage, which marks a key step for CAR-macrophage therapy to be applied in actual clinical practice.

CN115785289A discloses a chimeric antigen receptor, encoding gene, chimeric antigen receptor macrophage and use. The primary protein structure of the chimeric antigen receptor is sequentially: signal peptide, anti-HER2 single-chain antibody, hinge region, FCGR2A transmembrane region and FCGR2A intracellular region from the amino terminus to the carboxyl terminus. The chimeric antigen receptor macrophage expresses the chimeric antigen receptor as described above. The chimeric antigen receptor macrophage can effectively infiltrate into solid tumors, has a significant direct phagocytic killing effect on HER2 positive tumor cells, and can present tumor cell antigens to T cells to trigger the anti-tumor immune response of T cells.

Fibrosis is a chronic disease characterized by abnormal or excessive accumulation of extracellular matrix components in tissues and organs during the damage and repair response process. As the degree of fibrosis increases, this pathological process can affect almost any tissue and organ, and even lead to organ failure. Pulmonary fibrosis is a chronic, progressive interstitial lung disease. Pulmonary fibrosis seriously affects the respiratory function of the human body and can cause death due to complications such as acute deterioration of respiratory function, acute coronary syndrome, congestive heart failure, lung cancer, infection and venous thrombosis. According to statistics, there are about 3.4 million patients with pulmonary fibrosis in my country, and about 100,000 new cases are added each year. The median survival time of patients with idiopathic pulmonary fibrosis is 2-3 years, and the 5-year survival rate is less than 30%, which is lower than the survival rate of most cancers. Therefore, it is also called "cancer that is not cancer." There is a lack of effective treatment drugs for pulmonary fibrosis, especially idiopathic pulmonary fibrosis. The drugs currently approved by the FDA are mainly pirfenidone and nintedanib, but neither of them can prevent or reverse the progression of the disease, and may also cause adverse reactions such as nausea and diarrhea, making it difficult for patients to tolerate. When respiratory diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and pulmonary hypertension develop to the terminal stage, patients have difficulty breathing, and some even need oxygen 24 hours a day, cannot get out of bed to walk, and have a very low quality of life. Lung transplantation is the only treatment. However, due to age and comorbidity restrictions, lung transplantation is only suitable for a small number of patients, and lung transplantation is a highly difficult operation in the field of thoracic surgery.

Rejection refers to an immunological response of the human body to the donor, in which the transplant is attacked, destroyed, and cleared. Rejection of lung transplantation usually includes acute rejection and chronic rejection. Usually, there will be at least three acute rejections in the first three months after surgery, while chronic rejection occurs in most patients. Mild chronic rejection will not cause any harm to the patient's life, while severe rejection will require another lung transplant. The treatment for acute rejection includes routine maintenance therapy and shock therapy. The usual maintenance therapy is routine medication, and commonly used drugs include cyclosporine and hormones. Shock therapy is shock therapy with hormones for confirmed or highly suspected acute reactions. The treatment of chronic rejection is mainly accomplished by increasing immunosuppressants.

In summary, the development mechanism of pulmonary fibrosis is not yet fully understood, there is a lack of effective treatments, and the rejection reaction after lung transplantation also requires the development of new treatments. In addition, the contribution of different macrophage activation states to the progression and severity of the disease is still unclear, and its exact mechanism of action and cellular targets in fibrotic tissues and lung transplant rejection are also unclear. Therefore, how to develop a targeted CAR-macrophage and apply it to the treatment of fibrotic diseases, especially lung-related diseases, and the treatment of lung transplant-related rejection and other diseases has become one of the urgent problems to be solved in this field.

Summary of the invention

In order to solve the above technical problems, the present invention provides a chimeric antigen receptor targeting discoidin domain receptor 2, a chimeric antigen receptor cell, and a preparation method and application thereof. By preparing chimeric antigen receptor cells targeting discoidin domain receptor 2, especially chimeric antigen receptor macrophages targeting discoidin domain receptor 2, the macrophages are enhanced to treat fibrotic diseases, especially lung-related fibrotic diseases, and to treat lung transplant-related rejection and other diseases, which has broad application prospects.

To achieve this object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a chimeric antigen receptor targeting discoidin domain receptor 2, wherein the chimeric antigen receptor comprises an antigen recognition domain, a linker region, and an intracellular domain;

The antigen recognition domain includes a nanobody that recognizes discoidin domain receptor 2, the connection region includes a hinge region, a transmembrane domain and a TIR domain of a TLR4 protein, and the intracellular domain includes an intracellular domain of FcγR1.

Discoidin Domain Receptor 2 (DDR2) is a receptor-type protein tyrosine kinase that uses the extracellular matrix protein collagen as its ligand. In addition to its kinase function, DDR2 also promotes cell adhesion by activating β1-integrin. The unique function of DDR2 is to mediate the transmission of extracellular matrix signals into the cell, balance the regulation of the extracellular matrix, and participate in the regulation of cell growth, differentiation and metabolism.

Preferably, the Nanobody is derived from a heavy chain antibody.

Heavy chain antibodies refer to antibodies that lack light chains and consist only of heavy chains, and can come from antibodies produced by camelid species. Nanobodies refer to single-domain antibodies composed only of heavy chain variable regions obtained by cloning the variable regions of heavy chain antibodies, also known as VHH.

It is understandable that all Nanobodies with the function of recognizing DDR2 are applicable to the present invention, and the type of Nanobodies does not have a limiting effect on the present invention.

The following is an exemplary list of some Nanobodies with the function of recognizing DDR2, the amino acid sequence of the Nanobodies includes the sequences shown in SEQ ID NO.1-SEQ ID NO.5, the CDR amino acid sequence of SEQ ID NO.1 is shown in SEQ ID NO.6-SEQ ID NO.8, the CDR amino acid sequence of SEQ ID NO.2 is shown in SEQ ID NO.9-SEQ ID NO.11, the CDR amino acid sequence of SEQ ID NO.3 is shown in SEQ ID NO.12-SEQ ID NO.14, the CDR amino acid sequence of SEQ ID NO.4 is shown in SEQ ID NO.15-SEQ ID NO.17, and the CDR amino acid sequence of SEQ ID NO.5 is shown in SEQ ID NO.18-SEQ ID NO.20.

SEQ ID NO. 1: EVQLVESGGGLVQPGGSLRLSCAASGRIFSINVMGWFRQAPGKGRELV AAINTRGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASRKGYPSTRS DPYDYWGQGTQVTVSS.

SEQ ID NO. 2: EVQLVESGGGLVQPGGSLRLSCAASGITLSLYIMGWFRQAPGKGRELV AAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAADFYGNATLGY NYWGQGTQVTVSS.

SEQ ID NO.3: EVQLVESGGGLVQPGGSLRLSCAASGYILSPNVMGWFRQAPGKGREL VAAISWGDGRTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASRKGYPSTR SDPYNYWGQGTQVTVSS.

SEQ ID NO.4: EVQLVESGGGLVQPGGSLRLSCAASGYIFSIYAMGWFRQAPGKGRELV ARISWGDGGSTWYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAIKGRGVVT QLGYNYWGQGTQVTVSS.

SEQ ID NO.5: EVQLVESGGGLVQPGGSLRLSCAASGDTFSYYVMGWFRQAPGKGRE LVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAADPYISGTLG YNYWGQGTQVTVSS.

SEQ ID NO.6: GRIFSINV.

SEQ ID NO. 7: INTRGGST.

SEQ ID NO. 8: AASRKGYPSTRSDPYDY.

SEQ ID NO.9: GITLSLYI.

SEQ ID NO.10: ISRTGGST.

SEQ ID NO. 11: AADFYGNATLGYNY.

SEQ ID NO.12: GYILSPNV.

SEQ ID NO. 13: ISWGDGRT.

SEQ ID NO. 14: AASRKGYPSTRSDPYNY.

SEQ ID NO.15: GYIFSIYA.

SEQ ID NO. 16: ISWGDGST.

SEQ ID NO. 17: AAAIKGRGVVTQLGYNY.

SEQ ID NO. 18: GDTFSYYV.

SEQ ID NO. 19: ISRTGGST.

SEQ ID NO. 20: AADPYISGTLGYNY.

Preferably, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.21, including the antigen recognition domain, the hinge region, transmembrane domain and TIR domain of TLR4 protein, and the intracellular domain of FcγR1.

SEQ ID NO.21: MACQLDLLIGVIFMASPVLVISPCSSEVQLVESGGGLVQPGGSLRLSC AASGYILSPNVMGWFRQAPGKGRELVAAISWGDGRTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASRKGYPSTRSDPYNYWGQGTQVTVSSVIVVSTVAFLIYHFYFHLILIAGCKKYSRGESIYDAFVIYSSQNEDWVRNELVKNLEEGVPRFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSR WCIFEYEIAQTWQFLSSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDGKASNPEQTAEEEQETATWTKIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTPKEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS.

SEQ ID NO.21 of the present invention includes the nano antibody shown in SEQ ID NO.3. It can be understood that replacing the part of SEQ ID NO.21 that is identical to SEQ ID NO.3 with any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.5, or replacing it with any one of the nano antibodies having the function of recognizing DDR2, is within the scope of protection of the present invention.

In a second aspect, the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor targeting discoidin domain receptor 2 according to the first aspect.

In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

In a fourth aspect, the present invention provides a chimeric antigen receptor cell, wherein the chimeric antigen receptor cell expresses the chimeric antigen receptor targeting discoidin domain receptor 2 according to the first aspect.

Preferably, the initial cells of the chimeric antigen receptor cell include any one of macrophages, T cells, neutrophils or natural killer cells, or a combination of at least two of them.

Preferably, the macrophages include bone marrow-derived macrophages.

The CAR-macrophages prepared by the present invention can migrate to the fibrotic site in a targeted manner, eliminate DDR2 antigen-positive cells and extracellular matrix components, and successfully reverse the progression of fibrosis.

In a fifth aspect, the present invention provides a method for preparing a chimeric antigen receptor cell as described in the fourth aspect, the method for preparing a chimeric antigen receptor cell comprising: infecting initial cells with an adenovirus containing a coding sequence of a chimeric antigen receptor targeting discoidin domain receptor 2 to obtain a chimeric antigen receptor cell.

Preferably, the initial cells of the chimeric antigen receptor cell include any one of macrophages, T cells, neutrophils or natural killer cells, or a combination of at least two of them.

Preferably, the macrophages include bone marrow-derived macrophages.

In a sixth aspect, the present invention provides an immune cell preparation, comprising the chimeric antigen receptor cell described in the fourth aspect.

Preferably, the immune cell preparation further comprises a pharmaceutically acceptable excipient.

In a seventh aspect, the present invention provides use of the chimeric antigen receptor cell described in the fourth aspect and/or the immune cell preparation described in the sixth aspect in the preparation of a drug for treating fibrotic diseases.

Preferably, the fibrotic disease includes any one of pulmonary fibrosis, cardiac fibrosis, renal fibrosis or liver fibrosis, or a combination of at least two of them.

Preferably, the fibrotic disease includes mouse pulmonary fibrosis and/or human pulmonary fibrosis.

In an eighth aspect, the present invention provides use of the chimeric antigen receptor cell described in the fourth aspect and/or the immune cell preparation described in the sixth aspect in the preparation of a drug for treating lung transplant rejection.

Preferably, the lung transplant rejection reaction includes chronic allogeneic lung transplant dysfunction and/or acute rejection after lung transplantation.

Preferably, the lung transplant rejection reaction comprises chronic allogeneic lung transplant dysfunction in mice.

Preferably, the symptoms of chronic allogeneic lung transplant dysfunction include any one or a combination of at least two of alveolar destruction, bronchiolitis obliterans, peribronchial collagen deposition or pleural collagen deposition.

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

The present invention prepares chimeric antigen receptor cells targeting DDR2, preferably chimeric antigen receptor macrophages targeting DDR2, promotes M1 polarization of macrophages, significantly reduces the expression of fibrosis factors, specifically targets and phagocytizes DDR2 antigen-positive cells, and enhances the ability of macrophages to treat fibrotic diseases, especially lung-related fibrotic diseases, and lung transplant-related rejection and other diseases. The invention has high safety and broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the structure of adenoviral vectors of CAR molecules in CAR-macrophages;

FIG2 is a graph showing the expression ratio of CAR after adenovirus transfection of macrophages;

Figure 3 is a graph showing the secretion of different cytokines after macrophages were transduced with CAR and GFP control adenoviruses;

Figure 4 is a graph showing the results of non-specific phagocytosis of common latex beads by different macrophages, wherein Figure A is a flow cytometry result graph showing the phagocytosis of common red latex beads by different macrophages, and Figure B is a statistical graph showing the phagocytosis of common red latex beads by different macrophages;

Figure 5 is a diagram showing the results of using DDR2 positive beads to verify the targeted phagocytosis of CAR-macrophages, wherein Figure A is a confocal image of different macrophages engulfing DRR2 positive beads, with a scale bar of 10 μm, and Figure B is a statistical diagram of the number of different macrophages engulfing DDR2 positive beads;

FIG6 is a graph showing the results of the killing ability test of CAR-macrophages on DDR2-positive 293T cells;

FIG7 is a flowchart of the CAR-macrophage treatment of pulmonary fibrosis experiment;

Figure 8 is a graph showing changes in body weight of mice in each group after CAR-macrophage treatment;

Figure 9 is a diagram showing the results of Micro-CT evaluation of the efficacy of CAR-macrophages;

Figure 10 is a histopathological image showing the efficacy of CAR-macrophages;

Figure 11 is a graph showing the results of evaluating the efficacy of CAR-macrophages at the molecular expression level;

FIG12 is a graph showing the results of liver toxicity evaluation of CAR-macrophage therapy for pulmonary fibrosis;

FIG13 is a graph showing the evaluation results of CAR-macrophage therapy for pulmonary fibrosis and its toxicity to other organs;

FIG14 is a graph showing the expression of DDR2 in lung tissue of transplant rejection patients, wherein FIGA is a graph showing the mRNA level, and FIGB is a graph showing the protein level;

FIG15 is a flowchart of an experiment on CAR-macrophages for treating lung transplant rejection;

FIG16 is a diagram showing the effect of CAR-macrophages in treating lung transplant rejection;

FIG17 is a diagram showing the effect of CAR-macrophages on the pathological changes of lung transplant rejection;

FIG18 is a diagram showing the localization results of CAR-macrophages in a lung transplantation model;

FIG19 is a graph showing the change in the proportion of DDR2 in lung tissue slices of human pulmonary fibrosis patients treated with human CAR-macrophages;

Figure 20 is a graph showing the results of evaluating CAR-macrophage therapy for human pulmonary fibrosis at the molecular expression level.

DETAILED DESCRIPTION

To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

Example 1 Preparation of CAR-macrophages

Isolation, differentiation and culture of primary mouse bone marrow macrophages:

(1) Bone marrow isolation: The mice were euthanized, and the femur and tibia were separated using sterile forceps and scissors, and residual muscle or tissue attached to the bones was removed; a needle was inserted through the bottom of a 0.5 mL microcentrifuge tube to form a small hole, which was then placed in a larger 1.5 mL microcentrifuge tube; the femur was placed in a 0.5 mL microcentrifuge tube and centrifuged at 10,000 × g for 30 s to obtain bone marrow, and bone marrow mononuclear cells were obtained after red lysis.

(2) Differentiation into bone marrow-derived macrophages (BMDMs): Macrophage culture medium (high-glucose DMEM medium containing 10% fetal bovine serum) was supplemented with 50 ng/mL M-CSF, 12 × 10 ⁶ isolated total bone marrow mononuclear cells were suspended in 20 mL of macrophage culture medium, and the cells were cultured in a 15 cm non-tissue culture treated plate in a cell culture incubator at 37°C and 5% carbon dioxide for 5 days, and the growth of differentiated macrophages was confirmed under a microscope.

The nucleic acid sequence of (Ⅰ) DDR2 nanoantibody, (Ⅱ) nucleic acid sequence encoding hinge, transmembrane and TIR domains of TLR4, and (Ⅲ) nucleic acid sequence encoding intracellular domain of FcγR1 were integrated into a chimeric reading frame and inserted into Ad5-GFP adenovirus vector. The vector structure is shown in FIG1 .

The packaged adenovirus was concentrated to a virus titer of about 10 ¹⁰ PFU/mL and infected the prepared mouse macrophages with an infection MOI of 500. 48 hours after infection, the macrophage infection efficiency was detected by flow cytometry. Three independent infection experiments were performed, and the flow cytometry results showed that the adenovirus transfection efficiency exceeded 95%, as shown in Figure 2.

Uninfected mouse macrophages, referred to as wild macrophages, are GFP-negative and have no green fluorescence; Ad5-GFP adenovirus is a blank control virus that infects mouse macrophages, and the obtained cells are referred to as control macrophages, which are GFP-positive; Ad5-GFP adenovirus containing CAR molecules infects mouse macrophages, and the obtained cells are referred to as CAR-macrophages, which are GFP-positive.

Example 2 Verification of CAR-macrophages' ability to secrete cytokines, phagocytosis, and killing in vitro

(1) The expression of key M1 and M2 polarization genes in the wild-type macrophages, control macrophages, and CAR-macrophages obtained in Example 1 was detected by qRT-PCR technology. The results showed that, as shown in Figure 3, the expression of CAR-macrophages can effectively induce the M1 polarization phenotype and inhibit M2 polarization. The expression of IL-1β, IL-6, and iNOS cytokines was high, and the expression of CD206 was reduced.

(2) The nonspecific phagocytic effect of various macrophages on ordinary red latex beads was compared. There were 4 types of macrophages: wild macrophages (not infected with adenovirus), CAR-macrophages, lipopolysaccharide (LPS)-activated macrophages, and IL4+IL13 cytokine-activated macrophages. Different types of macrophages were incubated with ordinary red latex beads (latex beads: Sigma-Aldrich, L2778) at a ratio of 1:10 at 37°C for 4 h. The phagocytosis of macrophages was terminated with pre-cooled PBS. After removing excess magnetic beads, the red fluorescence ratio in macrophages was detected by flow cytometry to verify the phagocytic ratio of various BMDMs to red latex beads. The control group was wild macrophages that were not mixed with latex beads as a negative control. The results are shown in Figure 4. Figure 4A is a flow cytometry result of different macrophages phagocytizing ordinary red latex beads, and Figure 4B is a statistical chart of different macrophages phagocytizing ordinary red latex beads. The results show that the nonspecific phagocytic ability of CAR-macrophages is also stronger than that of other macrophages.

(3) The targeted specific phagocytosis of red latex beads coupled with DDR2 antigen by various macrophages was compared. Magnetic beads encapsulating the extracellular segment of DDR2 antigen were coupled with a Cy3 fluorescent group and incubated at 37°C for 4 h at a ratio of 5:1. Pre-cooled PBS was added to terminate the phagocytosis of macrophages, and the excess magnetic beads were removed before confocal microscopy. There were three types of macrophages: wild-type macrophages (not infected with adenovirus, stained with green fluorescent dye DIO for observation), control macrophages (Ad5-GFP blank control virus adenovirus, GFP positive), and CAR-macrophages (Ad5-GFP adenovirus containing CAR molecules, GFP positive). The results are shown in Figure 5, where Figure A is a confocal microscopy of different macrophages engulfing DRR2-positive beads, with a scale of 10 μm, and Figure B is a statistical diagram of the number of DDR2-positive beads engulfed by different macrophages. The engulfed magnetic beads were counted in 10 fields of view in each group. The results show that more CAR-macrophages can engulf DDR2-positive magnetic beads.

(4) Verification of the phagocytic effect of CAR-macrophages on 293T-DDR2 at the living cell level. CAR-macrophages and control macrophages have green fluorescence, while 293T-DDR2 has no fluorescence, as shown in the middle figure of Figure 6. After co-culturing 293T-DDR2: macrophages at a ratio of 5:1 for 24 hours, 293T-DDR2 was cultured alone as a control, and the cells were incubated at room temperature for 1 hour with a dose of 2 μg of ICG probe targeting DDR2 antigen, and then washed three times with PBS for fluorescence imaging, as shown in the left figure of Figure 6; and the fluorescence intensity was statistically analyzed, as shown in the right figure of Figure 6. In the imaging of ICG probe targeting DDR2 antigen, the fluorescence intensity of the three replicate wells co-cultured with 293T-DDR2 and CAR-macrophages was much lower than that of the control macrophage group. The results show that CAR-macrophages can target and kill DDR2 antigen-positive cells and have targeted phagocytic ability at the cellular level.

Example 3 Effect of CAR-macrophages in treating pulmonary fibrosis model

The bleomycin (BLM)-induced C57BL/6 mouse pulmonary fibrosis model is direct bronchial intubation, followed by instillation of BLM. The physiological structure of mouse lung tissue is one lobe on the left and four lobes on the right. The general modeling method is bilateral modeling, but the mortality rate is high, the incidence is inconsistent, and the uniformity evaluation effect is poor. Therefore, the present invention further optimizes the modeling method, by non-invasive unilateral intratracheal infusion of BLM, a dose of 40 μL of BLM (0.5 mg/mL, dissolved in sterile 0.9% saline), tracheal intubation to the left lobe of the mouse, inducing the establishment of a left pulmonary fibrosis model. The modeling mice were kept in a specific pathogen-free (SPF) facility. On the 8th day, the extent of the lesions in the left lung lobe was checked by in vivo Micro-CT scanning of small animals, and they were randomly divided into 4 groups, 1. Unmodeled mice, marked as untreated group; 2. BLM left modeling mice were injected with PBS through the tail vein; 3. BLM left modeling mice were injected with control macrophage group (Ad5-GFP blank control virus adenovirus) through the tail vein; 4. BLM left modeling mice were injected with CAR-macrophages through the tail vein. Cell dose: 5×10 ⁶ /mouse, the treatment time was 10 days after left modeling, and CT scan was performed again on the 27th day to check the lung condition of the mice, and the 28th day was the end of treatment. The specific experimental process is shown in Figure 7.

According to the changes in mouse weight in Figure 8, it can be concluded that BLM modeling in the left lung will cause severe weight loss in mice. After CAR-macrophage treatment on the 10th day, the weight of mice in the CAR-macrophage group gradually increased, and their weight was significantly higher than that of the PBS and control macrophage groups, and it was statistically significant.

As shown in the left figure of Figure 9, the CT results on the 27th day showed that the pulmonary parenchymal lesions caused by the left BLM modeling were alleviated in the CAR-macrophage group. As shown in the right figure of Figure 9, the yellow part represents the volume of the inflated lung. The larger the volume of the inflated lung, the better the pulmonary ventilation function. The PBS or control macrophage group of the left modeling on the 8th and 27th days showed a decrease in the volume of the inflated lung, but the CAR-macrophages greatly increased the volume of the inflated lung, indicating that the CAR-macrophages of the present invention can reverse fibrosis.

Lung tissue was collected for pathological analysis, Sirius red staining was performed, the scale bars were 1 mm and 100 μm, HE staining, α-smooth muscle actin (α-SMA) pathological section immunofluorescence, and DAPI staining of cell nuclei. As shown in Figure 10, Sirius red staining showed that the CAR-macrophage group showed a decrease in new collagen; HE staining showed that the alveolar structure in the left modeled lung lobe after CAR-macrophage treatment was more complete and the substantial lesions were reduced; α-SMA immunofluorescence staining also showed that the expression of α-SMA after CAR-macrophage treatment was significantly decreased.

To further detect the effects at the molecular level, the left lung lobe of the untreated group, PBS group, control macrophage group, and CAR-macrophage group after treatment was sampled, RNA was extracted, and reverse transcribed into cDNA. The qRT-PCR method was used to verify the expression levels of fibrosis factors collagen-1 (Collagen-1), fibronectin (Fibronectin), and DDR2.

As shown in Figure 11, the expression of fibrosis factors in the CAR-macrophage group was significantly reduced after treatment, which inhibited the progression of fibrosis at the molecular expression level, and the expression of DDR2 antigen was also reduced, further illustrating the targeted and specific phagocytic effect of CAR-macrophages.

The above experimental results all indicate that the M1-polarized CAR-macrophages targeting DDR2 in the present invention can effectively treat pulmonary fibrosis, opening up a new technical solution for the treatment of pulmonary fibrosis.

Example 4 Safety of CAR-macrophages

There was no death in mice with pulmonary fibrosis treated with CAR-macrophages, but it is necessary to evaluate possible pathological conditions in other organs. According to literature reports, CAR-macrophages are generally highly enriched in the liver, so the concentrations of serum alanine aminotransferase and aspartate aminotransferase were detected. As shown in Figure 12, the concentrations of alanine aminotransferase and aspartate aminotransferase in mice treated with CAR-macrophages did not increase, indicating that CAR-macrophages are not toxic to the liver. In addition, as shown in Figure 13, different organs were analyzed by HE staining, and no obvious abnormalities were found. Therefore, the M1-polarized DDR2-targeted CAR-macrophages obtained by the present invention have the potential to safely treat human pulmonary fibrosis.

Example 5 Effect of CAR-macrophages in the treatment of lung transplantation model

Lung transplantation (Ltx) is the only treatment option for patients with end-stage pulmonary fibrosis and other lung diseases such as COPD. However, Ltx often leads to chronic allograft dysfunction (CLAD), which is characterized by irreversible airway and lung parenchymal fibrosis. We first confirmed that the expression of DDR2 and collagen was elevated at the transcriptional and translational levels in lung tissues rejected by allograft patients, as shown in Figure 14, where A is the mRNA level and B is the protein level, indicating the presence of fibroblast activation and fibrosis.

The left lung of C57BL/10 (B10) mice was transplanted into C57BL/6 (B6) mice to establish a small allogeneic antigen-mismatched in situ Ltx model. Using this mouse Ltx model, the therapeutic effect of CAR-macrophages targeting DDR2 on CLAD was evaluated. The experimental process is shown in Figure 15. At the end of the experiment, the transplanted lung tissue on the left side was taken. It can be clearly seen by naked eye that the allogeneic transplanted lung of the PBS-treated recipient mouse was contracted and had signs of stiff fibrosis; and the allogeneic transplanted lung of the allogeneic mouse after CAR-macrophage treatment of the present invention was significantly closer to the left lung of a normal mouse, as shown in Figure 16.

The allogeneic transplanted lungs were stained with HE and Masson's staining, and the results are shown in Figure 17. The results of histological analysis showed that CAR-macrophage therapy significantly improved alveolar destruction, obliterative bronchiolitis, and peribronchial and pleural collagen deposition in the allogeneic transplanted lungs. The results of Masson's staining also showed that the collagen content decreased significantly after CAR-macrophage therapy.

To demonstrate the chemotaxis of CAR-macrophages in allograft lungs, a control macrophage group (Ad5-GFP blank control virus adenovirus) and CAR-macrophages labeled with a red cell membrane fluorescent probe (DID fluorescence) were injected into Ltx mice, and the DID signals of all lung lobes were analyzed on day 28. As shown in Figure 18, DID fluorescence-labeled CAR-macrophages were preferentially enriched in the left lung of allografts, which means that CAR-macrophages show specific chemotaxis targeting DDR2 in allograft lungs, laying the foundation for anti-fibrosis and protective effects in allograft lung transplantation.

Example 6 Role of CAR-macrophages in the treatment of human pulmonary fibrosis

According to the aforementioned embodiments, in the specific models of mouse unilateral pulmonary fibrosis and mouse LTx, the CAR-macrophages of the present invention can migrate to the fibrotic site in a targeted manner, eliminate DDR2 antigen-positive cells and extracellular matrix components, and successfully reverse the progression of fibrosis.

In order to verify the effect of the CAR-macrophages provided by the present invention in treating human pulmonary fibrosis, a human CAR vector was constructed, human macrophages were infected, and the effect of human M1 polarized macrophages targeting DDR2 in treating human pulmonary fibrosis was studied.

The fibrotic lung tissue of patients with pulmonary fibrosis undergoing lung transplantation was filled with agar and cut into 600 μm thick slices using an oscillating slicer. The slices were divided into three groups: an untreated group, a control macrophage group (Ad5-GFP blank control virus adenovirus) and a human CAR-macrophage group. After co-culture for 5 days, samples were collected for fluorescence imaging of DDR2 antigens and QRT-PCR to measure changes in pulmonary fibrosis indicators such as α-SMA, collagen (Collagen-I) and fibronectin (Fibronectin).

As shown in Figure 19, compared with the slices co-cultured with human control macrophage group cells, the DDR2 level in the slices co-cultured with human CAR-macrophages was much lower, indicating that human CAR-macrophages targeted and eliminated DDR2-positive cells. As shown in Figure 20, the transcription levels of genes encoding α-SMA, collagen, and fibronectin were all inhibited by human CAR-macrophages. These results further confirm that the CAR design provided by the present invention and the CAR-macrophages that target DDR2 and can polarize macrophages to M1 can also be used for human anti-fibrosis treatment.

In summary, the present invention prepares chimeric antigen receptor cells targeting DDR2, preferably chimeric antigen receptor macrophages targeting DDR2, promotes M1 polarization of macrophages, significantly reduces the expression of fibrosis factors, specifically targets and phagocytizes DDR2 antigen-positive cells, and enhances the ability of macrophages to treat fibrotic diseases, especially lung-related fibrotic diseases, and treat lung transplant-related rejection and other diseases. It has high safety and broad application prospects.

The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims (10)

1. A chimeric antigen receptor targeting discoidin domain receptor 2, characterized in that the chimeric antigen receptor comprises an antigen recognition domain, a connecting region and an intracellular domain; The antigen recognition domain includes a nanobody that recognizes discoidin domain receptor 2, the connection region includes a hinge region, a transmembrane domain and a TIR domain of a TLR4 protein, and the intracellular domain includes an intracellular domain of FcγR1. 2. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the chimeric antigen receptor targeting discoidin domain receptor 2 according to claim 1. 3. A recombinant vector, characterized in that the recombinant vector contains the nucleic acid molecule according to claim 2. 4. A chimeric antigen receptor cell, characterized in that the chimeric antigen receptor cell expresses the chimeric antigen receptor targeting discoidin domain receptor 2 according to claim 1; Preferably, the initial cells of the chimeric antigen receptor cell include any one or a combination of at least two of macrophages, T cells, neutrophils or natural killer cells; Preferably, the macrophages include bone marrow-derived macrophages. 5. The method for preparing a chimeric antigen receptor cell according to claim 4, characterized in that the method for preparing a chimeric antigen receptor cell comprises: infecting an initial cell with an adenovirus containing a coding sequence of a chimeric antigen receptor targeting discoidin domain receptor 2 to obtain a chimeric antigen receptor cell; Preferably, the initial cells of the chimeric antigen receptor cell include any one or a combination of at least two of macrophages, T cells, neutrophils or natural killer cells; Preferably, the macrophages include bone marrow-derived macrophages. 6. An immune cell preparation, characterized in that the immune cell preparation comprises the chimeric antigen receptor cell according to claim 4; Preferably, the immune cell preparation further comprises a pharmaceutically acceptable excipient. 7. Use of the chimeric antigen receptor cell according to claim 4 and/or the immune cell preparation according to claim 6 in the preparation of a drug for treating fibrotic diseases. 8. The use according to claim 7, characterized in that the fibrosis disease comprises any one of pulmonary fibrosis, cardiac fibrosis, renal fibrosis or liver fibrosis or a combination of at least two thereof; Preferably, the fibrotic disease includes mouse pulmonary fibrosis and/or human pulmonary fibrosis. 9. Use of the chimeric antigen receptor cell according to claim 4 and/or the immune cell preparation according to claim 6 in the preparation of a medicament for treating lung transplant rejection. 10. The use according to claim 9, characterized in that the lung transplant rejection reaction includes chronic allogeneic lung transplant dysfunction and/or acute rejection after lung transplantation; Preferably, the lung transplant rejection reaction includes chronic allogeneic lung transplant dysfunction in mice; Preferably, the symptoms of chronic allogeneic lung transplant dysfunction include any one or a combination of at least two of alveolar destruction, bronchiolitis obliterans, peribronchial collagen deposition or pleural collagen deposition.