Chimeric antigen receptor-macrophage targeting HSP70 and application thereof in treatment of noise-induced hearing loss

Abstract

The invention discloses an HSP70-targeting chimeric antigen receptor-macrophage and application thereof in treatment of noise-induced hearing loss, an extracellular domain of the HSP70-targeting chimeric antigen receptor comprises an HSP70 specific binding fragment, an intracellular domain comprises an IL-4 signal channel functional element, a signal switch type CAR structure of the HSP70 is formed, and the macrophage can be used for treating noise-induced hearing loss, so that the HSP70-targeting chimeric antigen receptor-macrophage can be used for treating noise-induced hearing loss. The HSP70-targeted chimeric antigen receptor-macrophage can be used for preparing the HSP70-targeted chimeric antigen receptor-macrophage, is used for mediating polarization of the macrophage to an M2 type and exerting an anti-inflammatory function, is proved to be capable of specifically responding to an inner ear inflammation microenvironment, switching an anti-inflammatory phenotype and protecting hair cells and auditory nerves, can be used for treating noise-induced hearing loss, and can be used for preparing the HSP70-targeted chimeric antigen receptor-macrophage. The limitation that traditional hearing-aid equipment supports treatment on symptoms is broken through, and the hearing-aid device has a remarkable application prospect.

Description

Technical Field

[0001] This invention relates to the field of deafness treatment technology, specifically to chimeric antigen receptor-macrophages targeting HSP70 and their application in the treatment of noise-induced hearing loss. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved significant success in the treatment of tumors such as lymphoma. It primarily utilizes gene editing technology to equip the patient's own T cells with a CAR system, enabling them to precisely recognize specific antigens on the surface of cancer cells. The modified CAR-T cells are then infused into the patient, achieving a "precision strike" against cancer cells. In recent years, this technology has expanded to the fields of natural killer cells and macrophages. Chimeric antigen receptor macrophage (CAR-M) therapy, as an innovative treatment strategy, demonstrates unique therapeutic potential. It specifically recognizes tumor-associated antigens, mediating efficient phagocytosis to eliminate tumor cells. Macrophages are widely distributed throughout the body and can be divided into M1 and M2 types. In addition to the phagocytic function required for anti-tumor activity, they also possess regenerative and inflammatory regulatory functions, making them ideal candidates for cell therapy. M1 macrophages produce high levels of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, exhibiting strong phagocytic and tumor cell-killing capabilities—the main mechanism underlying the widely used CAR-M therapy for solid tumors. M2 macrophages, on the other hand, produce anti-inflammatory cytokines, such as IL-10 and TGF-β, participating in inflammation suppression, tissue repair, and angiogenesis promotion. Infusion of anti-inflammatory M2 macrophages has shown damage-relieving effects in various non-tumor disease models. However, traditional strategies for regulating macrophage phenotypes suffer from insufficient targeting and susceptibility to interference from the inflammatory microenvironment, making it difficult to precisely and stably exert anti-inflammatory effects. CAR-M, by targeting and activating "signal switch" antigens, can precisely regulate macrophage polarization towards M2 anti-inflammatory macrophages. This therapeutic approach has been explored and applied in non-tumor fields such as autoimmune diseases and fibrotic diseases with promising results.

[0003] Damage to the sensory epithelium and auditory nerve in the inner ear is irreversible, and current clinical interventions primarily rely on hearing aids or cochlear implants to assist hearing. On the one hand, these assistive devices struggle to accurately reproduce the natural listening experience and cannot meet the auditory needs in complex scenarios; on the other hand, children with congenital deafness are highly susceptible to missing the critical window for speech development, impacting their subsequent language abilities and social function. Therefore, targeted treatment in the early stages of damage has become an important direction for improving patient prognosis and filling clinical gaps. Recent studies have shown that inner ear inflammation and immunity are key mechanisms for maintaining auditory and balance homeostasis. The inner ear immune response to acoustic trauma, ototoxic drugs, and other stressors directly determines the degree of damage and the effectiveness of repair. With the development of electronic technology, non-occupational noise generated by transportation, communication, and entertainment in daily life is increasingly affecting people's hearing. WHO statistics indicate that approximately 16% of acquired hearing loss in adults worldwide is caused by noise exposure. Currently, treatment mainly relies on symptomatic supportive care and hearing aids; there are currently no clinically approved drugs for treating diseases related to noise exposure. After noise exposure, resident macrophages in the inner ear are rapidly activated, while circulating immune cells infiltrate damaged sites such as the spiral ligament and basement membrane. Overactivation can release pro-inflammatory factors such as TNF-α and IL-1β, which can aggravate hair cell damage. In addition, increasing evidence suggests that the whole immune system can also "hear" noise. Noise can cause a rise in various inflammatory markers through neuroimmune responses, oxidative stress, and other pathways.

[0004] Our team's research found that heat shock protein 70 (HSP70) expression increases during the early stages of noise exposure, specifically during the temporary threshold shifts (TSS) phase, mediating protection of the inner ear from noise damage. Strengthening this mechanism during this period can prolong the temporary threshold shift phase of NIHL and prevent progression to permanent threshold shifts (PTS), thereby avoiding irreversible hearing loss. Summary of the Invention

[0005] This invention provides a "signal-switching" CAR-M cell therapy targeting the noise-induced hearing loss target HSP70, which converts inflammatory signals into anti-inflammatory signals and aims to salvage noise-induced hearing loss through systemic infusion of modified CAR-M.

[0006] In view of this, the solution of the present invention is as follows: A first aspect of the present invention is to provide a chimeric antigen receptor targeting HSP70, comprising a single-chain antibody scFv that recognizes human HSP70 antigen, a CD8α hinge region and a transmembrane domain, and an IL-4Rα intracellular co-stimulatory signal transduction domain; the amino acid sequence of the single-chain antibody scFv that recognizes human HSP70 antigen is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequences of the CD8α hinge region and transmembrane domain are shown in SEQ ID NO.2.

[0008] Furthermore, the amino acid sequence of the intracellular co-stimulatory signal transduction structure of the IL-4Rα is shown in SEQ ID NO.3.

[0009] Furthermore, the chimeric antigen receptor also includes a fluorescent tag, preferably a green fluorescent protein (GFP) tag.

[0010] Furthermore, the chimeric antigen receptor also includes a signal peptide, which is a granulocyte-macrophage colony-stimulating factor.

[0011] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.5.

[0012] A second aspect of the invention is to propose a gene encoding the chimeric antigen receptor described in the first aspect.

[0013] A third aspect of the present invention is to provide a recombinant vector comprising the gene described in the second aspect; preferably, the recombinant vector is a lentiviral vector.

[0014] A fourth aspect of the invention is to provide a chimeric antigen receptor-macrophage that expresses a chimeric antigen receptor targeting HSP70 as described in the first aspect.

[0015] Furthermore, the chimeric antigen receptor-macrophage is an M2 type macrophage.

[0016] A fifth aspect of the invention is the application of the chimeric antigen receptor-macrophage described in the fourth aspect in the preparation of a medicament for the treatment of noise-induced hearing loss.

[0017] Furthermore, the drug is an injectable preparation, preferably for infusion into the inner ear.

[0018] Furthermore, the drug includes any one of the following uses: 1) Improve hearing frequency thresholds; 2) Reduce pro-inflammatory cytokines, including IL-1β, TNFα and IL-6.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The extracellular domain of the chimeric antigen receptor targeting HSP70 provided by the present invention contains an HSP70-specific binding fragment, and the intracellular domain contains IL-4 signaling pathway functional elements, forming a "signal switch" CAR structure targeting HSP70. It can be used to prepare chimeric antigen receptor-macrophages targeting HSP70 and to mediate macrophage polarization to M2 type and exert anti-inflammatory function.

[0020] The chimeric antigen receptor-macrophage targeting HSP70 described in this invention has been verified to specifically respond to the inflammatory microenvironment of the inner ear, switch anti-inflammatory phenotypes, and protect hair cells and auditory nerves. It can be used to treat noise-induced hearing loss, breaking through the limitations of traditional symptomatic supportive treatment (hearing aids) and showing significant potential for drug therapy applications.

[0021] The chimeric antigen receptor-macrophage targeting HSP70 described in this invention directly converts HSP70-mediated pro-inflammatory signals into anti-inflammatory signals, enabling macrophages to polarize to the M2 type. After the inflammation subsides, the CAR-M effect automatically terminates, avoiding immunosuppression or abnormal tissue repair caused by long-term anti-inflammatory treatment.

[0022] The chimeric antigen receptor-macrophage targeting HSP70 described in this invention can be infused into the inner ear as a drug, precisely targeting damaged sites such as the spiral ligament and basilar membrane, protecting hair cells and the auditory nerve, and blocking the progression of damage in the early stages of hearing loss. Addressing the pathological characteristics of noise-induced neuroimmune responses and elevated systemic inflammatory markers, systemic infusion achieves simultaneous improvement of local and systemic inflammation in the inner ear, overcoming the limitations of traditional treatments that only focus on the inner ear and expanding the application boundaries of CAR-M therapy. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the chimeric antigen receptor structure targeting HSP70 described in one embodiment of the present invention.

[0024] Figure 2 This is the verification result of the polarization phenotype and transfection efficiency of macrophages after transfecting the CAR vector targeting HSP70 in Example 1 of the present invention.

[0025] Figure 3 The results of HSP70-targeted CAR-M in Example 2 of this invention show the improvement of hearing and inhibition of pro-inflammatory cytokines in a noise-induced hearing loss model. Detailed Implementation

[0026] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In one embodiment, a CAR targeting HSP70 is proposed. The core design of this CAR utilizes a "signal transduction" mechanism to convert inflammatory signals into anti-inflammatory signals, thereby regulating the macrophage phenotype. Its structure comprises three key functional domains that synergistically achieve targeted recognition and signal transduction, as detailed below: 1) Extracellular recognition domain: The anti-HSP70 single-chain variable fragment (scFv) is used to specifically bind to HSP70 in the inflammatory microenvironment, ensuring that CAR is activated only at the site of inflammation.

[0028] The anti-HSP70 scFv sequence (SEQ ID NO.1) is as follows: MAEVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKRAAA 2) Transmembrane connection domain: Using the hinge region and transmembrane domain of mouse or human CD8α, it is responsible for connecting the extracellular domain and the intracellular domain to maintain the stability of the CAR structure.

[0029] The transmembrane linker domain sequence (SEQ ID NO.2) is: GLDFACDIYIWAPLAG.

[0030] 3) Intracellular signaling domain: Integrates the intracellular domain of interleukin-4 receptor α (IL-4Rα). When scFv binds to HSP70, it activates downstream IL-4 signaling pathways (such as p-STAT6 and p-AKT) through this domain, inducing macrophages to transform into an anti-inflammatory phenotype (M2 type) instead of initiating conventional pro-inflammatory signals.

[0031] The intracellular signal domain sequence (SEQ ID NO.3) is as follows: KIKKIWWDQIPTPARSPLVAIIIQDAQVPLWDKQTRSQESTKYPHWKTCLDKLLPCLLKHRVKKKTDFPKAAPTKSLQSPGKAGWCPMEVSRTVLWPENVSVSVVRCMELFEAPVQNVEEEEDEIVKEDLSMSPENSG GCGFQESQADIMARLTENLFSDLLEAENGGLGQSALAESCSPLPSGSGQASVSWACLPMGPSEEATCQVTEQPSHPGPLSGSPAQSAPTLACTQVPLVLADNPAYRSFSDCCSPAPNPGELAPEQQQADHLEEEEPPS PADPHSSGPMQPVESWEQILHMSVLQHGAAAGSTPAPAGGYQEFVQAVKQGAAQDPGVPGVRPSGDPGYKAFSSLLSSNGIRGDTAAAGTDDGHGGYKPFQNPVPNQSPSSVPLFTFGLDTELSPSPLNSDPPKSPP ECLGLELGLKGGDWVKAPPPADQVPKPFGDDLGFGIVYSSLTCHLCGHLKQHHSQEEGGQSPIVASPGCGCCYDDRSPSLGSLSGALESCPEGIPPEANLMSAPKTPSNLSGEGKGPGHSPVPSQTTEVPVGALGIAVS 1. Design and synthesis of scFv: The anti-HSP70 scFv is constructed based on the heavy chain (VH) and light chain (VL) variable regions of the HSP70 binding antibody to ensure its specific binding ability to HSP70.

[0032] 2. Assembly of the full-length CAR sequence: The CAR was assembled in the order of “labeled peptide-extracellular domain-transmembrane domain-intracellular domain”, and the hinge region and transmembrane domain of anti-HSP70scFv and CD8α (connection function) and the intracellular domain of IL-4Rα (signal transduction) were sequentially linked; green fluorescent protein GFP was introduced as a marker for transduction efficiency detection (SEQ ID NO.4).

[0033] The labeled peptide is granulocyte-macrophage colony-stimulating factor GM-CSF–2A, which is used to maintain the functional plasticity of macrophages.

[0034] A schematic diagram of the assembled CAR is shown below. Figure 1 As shown, the full-length amino acid sequence is shown in SEQ ID NO.5.

[0035] Example 1

[0036] I. Design and Construction of CAR

[0037] The core design of CARs (autoimmune chemoradiotransfer) is to convert inflammatory signals into anti-inflammatory signals through a "signal transduction" mechanism, thereby regulating the macrophage phenotype. Its structure contains three key functional domains that work synergistically to achieve target recognition and signal transduction, as detailed below: 1. Anti-HSP70-CAR-M construction scheme (1) CAR sequence design: The extracellular recognition domain anti-HSP70-scFv was constructed based on the variable regions of the heavy chain (VH) and light chain (VL) of the HSP70 binding antibody (GenBank: QKZ93314.1). The anti-HSP70 scFv sequence is shown in SEQ ID NO.1. The gene fragment was synthesized by Priscilla Technology Co., Ltd.

[0038] Transmembrane connection domain: Using the hinge region and transmembrane domain of mouse CD8α, it is responsible for connecting the extracellular domain and the intracellular domain, maintaining the stability of the CAR structure.

[0039] Intracellular signaling domain: This domain integrates the intracellular domain of interleukin-4 receptor α (IL-4Rα). When scFv binds to HSP70, it activates downstream IL-4 signaling pathways (such as p-STAT6 and p-AKT) through this domain, inducing macrophages to transform into an anti-inflammatory phenotype (M2 type), rather than initiating conventional pro-inflammatory signaling. A truncated CAR (CARΔ) was constructed as a control, lacking the overexpressed fragment, to verify the necessity of the signaling domain.

[0040] The constructed anti-HSP70-CAR amino acid sequence is shown in SEQ ID NO.5.

[0041] 2. Lentiviral packaging

[0042] (1) Plasmid preparation: After the construction of the recombinant lentiviral vector is completed, a large amount of plasmid is extracted to prepare for subsequent transfection.

[0043] (2) 293T cell culture and transfection: Seed 293T cells into 10cm dishes and control the cell density to 70-80% the next day for transfection (ensure that the cells are passaged no more than 20 times and are in good condition). Remove the culture dish 1 hour before transfection, remove the original culture medium, and add 10ml of Opti-MEM medium.

[0044] (3) Preparation of transfection complex: Dissolve 32 μg of plasmid to be transfected (packaging plasmid: shuttle plasmid = 1:1) in Opti-MEM medium, total volume 500 μl, mix gently and let stand for 5 min; dissolve the transfection reagent in Opti-MEM medium, total volume 500 μl, mix gently and let stand for 5 min; add the dilution of the transfection reagent dropwise to the dilution of the plasmid, mixing gently while adding, and let stand at room temperature for 20 min to allow the DNA to fully combine with the transfection reagent to form a stable transfection complex.

[0045] (4) Transfection and medium change: Take out the culture dish and add the prepared DNA-transfection reagent mixture; after 6 hours of transfection, remove the culture medium, wash once with PBS, and add 10 ml of fresh complete culture medium to continue culturing.

[0046] (5) Lentiviral harvest: Collect the supernatant of 293T cells 48h and 72h after transfection, and aliquot them into 50ml centrifuge tubes; centrifuge at 3500rpm at room temperature for 10min to remove cells and large debris; filter the supernatant into an ultracentrifuge tube using a 0.45μm filter membrane.

[0047] (6) Lentiviral concentration: Centrifuge the above supernatant at 30,000 rpm and 4°C for 2 h. A white virus precipitate will be visible on one side of the tube wall. Discard the supernatant and invert the centrifuge tube onto sterile absorbent paper to remove any remaining supernatant. Add 80 μl to 120 μl of DPBS according to the amount of precipitate. Seal the tube opening with sealing film and dissolve the precipitate overnight at 4°C. Aliquot the virus as needed and store it in a -80°C refrigerator.

[0048] (7) Lentiviral titer determination: ① The day before infection, administer 1×10 mg per well. 5 24-well plates were seeded at a density of 1,000 cells.

[0049] ② Viral infection: Add 10 μl, 1 μl, and 0.1 μl of virus to different cell culture wells (without adding Polybrene). The virus can be diluted before adding.

[0050] ③ Change the culture medium: After 24 hours of infection, replace the culture medium with fresh medium and continue culturing.

[0051] ④ Cell treatment: After 72-96 hours, discard the cell supernatant, collect the cells and extract genomic DNA from 293T cells.

[0052] ⑤qPCR detection: Using the extracted genome as a template, the internal reference gene or viral sequence WPRE is quantitatively detected.

[0053] ⑥ Plotting the standard curve: The amplification efficiency of the standard curve should be between 0.9 and 1.1, and R0 should be within the range of 0.9-1.1. 2 If the value is >0.990, primer specificity needs to be tested and the standard rediluted.

[0054] ⑦ Titer Calculation: Calculate the lentivirus titer (TU / ml) using the following formula: Lentiviral titer (TU / ml) = (q-PCR copies / ul × M × gDNA dilution factor) × 1000ml / virus volume of infected cell (ul).

[0055] M represents the elution volume after gDNA extraction, and 1000 indicates that the viral units are converted from μl to ml.

[0056] II. Extraction, differentiation, polarization, and CAR lentiviral transduction of BMDMs

[0057] 1. Bone marrow-derived macrophages (BMDMs) were prepared using existing methods. Cell differentiation was detected by F4 / 80 staining on day 6 to confirm successful differentiation of BMDMs.

[0058] 2. Transduction and culture: Four days after BMDMs differentiated, the cells were transduced with concentrated CAR lentivirus at a multiplicity of infection of 500 infection units / cell. After transduction, 20 ng / ml macrophage colony-stimulating factor (M-CSF) was added and the cells were cultured for another 2 days to complete the preparation of CAR-modified macrophages.

[0059] like Figure 2 As shown, the morphological characteristics of macrophage phenotypic polarization: Bone marrow-derived macrophages (BMDM) transfected with LV-anti-HSP70-CAR-GFP can polarize from the M0 phenotype to spindle-shaped, elongated M2 macrophages, whose morphology is highly consistent with IL-4-induced M2 BMDM (BMDM+IL-4). Figure 2 A, 2B).

[0060] III. Flow cytometry analysis of post-polarized macrophage function

[0061] 1. Wash once with pre-cooled PBS containing 2% FBS, add 300ul of trypsin (6-well plate) and digest at 37°C for 5min. Add 500ul of PBS containing 2% FBS to stop digestion. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000rpm for 5min.

[0062] 2. Wash once with 2% FBS in PBS buffer, centrifuge at 1500 rpm for 5 min. Resuspend cells in 2% FBS PBS and count them, adjusting the cell concentration to 1-2 × 10⁻⁶ cells / mL. 7 cells / mL (make sure the concentration is appropriate).

[0063] 3. Take 100 µL of cell suspension (approximately 1-2 × 10⁻⁶ cells / mL). 6Add cells to a flow cytometry tube. Add pre-diluted LIVE / DEAD dye (dilute according to instructions), mix gently, incubate on ice in the dark for 10 minutes, add 500 μL of 2% FBS PBS to stop staining, centrifuge and discard the supernatant.

[0064] 4. Fc receptor blocking: Resuspend cells in 100 μL PBS, add 0.5 μL Fc Block (CD16 / 32 antibody, 0.5-1 μg / 100 μL), mix gently, and incubate on ice for 15 minutes.

[0065] 5. Surface antibody incubation: No washing is required. Directly add fluorescent antibodies against the surface markers CD11b / CD38 / CD86 / EGR2. Gently mix and incubate on ice in the dark for 30 minutes. After incubation, wash the cells with 1 mL of PBS buffer, centrifuge, and discard the supernatant.

[0066] 6. Cell fixation and permeabilization: Add 500 μL of 4% PFA to fix the cells, incubate at room temperature in the dark for 20 min, add 500 μL of PBS, centrifuge at 3000 rpm for 5 min and discard the supernatant. Add 500 μL of 0.1% Triton X-100 for permeabilization for 5 min, and gently vortex to mix.

[0067] 7. Wash the cells once with buffer, then stain the cells with anti-CD206 antibody diluted in PBS overnight.

[0068] 8. Washing and Analytical Processing: Wash cells twice with PBS buffer and resuspend cells in 100 μL of PBS buffer. Keep sample tubes on ice and protected from light before analysis.

[0069] Flow cytometry analysis of CAR vector transfection efficiency and macrophage phenotype verification showed that the transfection efficiency of LV-anti-HSP70-CAR-GFP for BMDM was approximately 37.3%. Figure 2 C), indicating that the vector can effectively transfect target cells; further flow cytometry analysis showed that the mean fluorescence intensity (MFI) of CD206, the M2 macrophage-specific marker on the surface of BMDM, was significantly increased after transfection, while no such change was observed in the empty CAR-M (ΔCAR-M) treatment group, confirming that anti-HSP70-CAR can mediate the polarization of BMDM towards the M2 phenotype with anti-inflammatory activity. Figure 2 D、 Figure 2 E).

[0070] Example 2

[0071] 1. Construction of a mouse model of noise-induced hearing loss

[0072] Based on the noise-induced hearing loss modeling conditions previously established by our team (Xiong Xiao, Sun Yu. Cochlear macrophages participate in the occurrence of noise-induced hearing loss [D]. Huazhong University of Science and Technology, 2020. DOI:10.27157 / d.cnki.ghzku.2020.003814.), 6-week-old wild-type (WT) mice were placed in specially designed square iron cages. The cages were surrounded by wire to ensure good ventilation and sound transmission, with the sound intensity range on the maximum diagonal not differing by more than 2 dB. The mice were allowed to move freely within the cages. A speaker was placed on the iron cages, with the other end connected to a computer. Specific software generated, amplified, and transmitted the sound to the speaker. The iron cages were placed in a sealed, soundproof chamber. The computer software was adjusted to produce broadband noise (BBN) with a center frequency of 250 Hz-16 kHz at 110 dBSPL. The noise was transmitted to the speaker located on the other end of the iron cages, causing noise exposure in the mice. The noise exposure time for each group of mice was 2 hours.

[0073] 2. Cell infusion treatment

[0074] Model Grouping and Treatment: After the noise-induced hearing loss (NIHL) model was successfully constructed (i.e., 72 hours after noise exposure), the experimental animals were divided into three groups and treated as follows: 1) NIHL+CAR-M treatment group: CAR-M was infused via two different methods, with the tail vein infusion dose being 1×10⁻⁶. 6 Each individual, administered via tympanic membrane infusion (through tympanic membrane puncture) at a dose of 5 × 10⁻⁶ units / animal. 4 One / each; 2) NIHL model control group: Infused with an equal amount of unmodified macrophages; 3) Normal hearing control group: Infused with an equal volume of physiological saline.

[0075] The specific operation timeline is shown in Table 1.

[0076] Table 1: NIHL Treatment Timeline

[0077] Note: P represents the number of days after the mouse is born, and the same applies below.

[0078] 2. Setting detection time points: Accurately record the cell infusion time and set 1 day (early stage), 7 days (mid stage), and 28 days (long stage) after infusion as subsequent detection time points.

[0079] Verification of the effect of anti-HSP70-CAR-M on hearing improvement in NIHL mice: After intravenous injection of anti-HSP70-CAR-M into noise-exposed NIHL model mice, auditory brainstem response (ABR) tests at 3 days showed that, compared with the uninjected NIHL model group and the wild-type (WT) group, the injected group mice exhibited significant improvement in hearing thresholds across the entire frequency range from 8 kHz to 40 kHz, with a recovery range of 10–25 days of hearing threshold (dBSPL). Figure 3 A).

[0080] 3. Site-specific distribution detection

[0081] Sample collection: Three mice were selected from each group at each testing time point and sacrificed by cervical dislocation to collect cochlear tissue samples.

[0082] 4. Immunofluorescence detection: Collected cochlear tissue was prepared into frozen sections, observed and counted under a laser confocal microscope, and the distribution of GFP-positive cells was analyzed in the spiral ligament and spiral ganglion. For example... Figure 2 As shown in F, CAR-M precisely targets the spiral ligament and spiral ganglion.

[0083] Verification of the regulatory effect of anti-HSP70-CAR-M on cochlear inflammatory factors: qPCR detection of RNA in the cochlear tissues of mice in each group revealed that, compared with the uninjected NIHL model group, the mRNA expression levels of pro-inflammatory cytokines IL-1β, TNFα, and IL-6 in the anti-HSP70-CAR-M injection group were significantly reduced. Figure 3 B, 3C, 3D).

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A chimeric antigen receptor targeting HSP70, characterized in that, It includes a single-chain antibody scFv that recognizes the human HSP70 antigen, a CD8α hinge region and a transmembrane domain, and an IL-4Rα intracellular co-stimulatory signal transduction domain; the amino acid sequence of the single-chain antibody scFv that recognizes the human HSP70 antigen is shown in SEQ ID NO.

1.

2. The chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequences of the CD8α hinge region and transmembrane domain are shown in SEQ ID NO.2; And / or, the amino acid sequence of the intracellular co-stimulatory signal transduction structure of the IL-4Rα is shown in SEQ ID NO.3; And / or, the chimeric antigen receptor further includes a fluorescent tag.

3. The chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor also includes a signal peptide, which is a granulocyte-macrophage colony-stimulating factor.

4. The chimeric antigen receptor according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

5.

5. The gene encoding the chimeric antigen receptor as described in any one of claims 1-4.

6. A recombinant vector, characterized in that, It contains the gene described in claim 5.

7. A chimeric antigen receptor-macrophage, characterized in that, It expresses the chimeric antigen receptor targeting HSP70 as described in any one of claims 1-4.

8. The chimeric antigen receptor-macrophage according to claim 7, characterized in that, It is an M2 type macrophage.

9. The use of the chimeric antigen receptor-macrophage as described in claim 7 or 8 in the preparation of a drug for treating noise-induced hearing loss.

10. The application according to claim 9, characterized in that, The drug has any of the following uses: 1) Improve hearing frequency thresholds; 2) Reduce pro-inflammatory cytokines, including IL-1β, TNFα and IL-6.

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