A method for preparing CART cells and its application in anti-tumor treatment

Abstract

The present invention discloses a HER2 single-domain antibody-pH responsive targeting polypeptide fusion structure and its application in CART therapy. The fusion structure combines a pH-sensitive targeting motif with a high-affinity HER2 single-domain antibody, maintains a hidden state at neutral pH, reduces nonspecific binding, and undergoes conformational changes in the tumor microenvironment (pH <6.8), exposing the HER2 targeting motif, thereby significantly improving the HER2-positive tumor recognition and killing ability of CART cells, reducing off-target toxicity, and improving efficacy. Therefore, the present invention combines a pH-responsive targeting polypeptide with a HER2 single-domain antibody, improves the HER2-positive tumor targeting and anti-tumor activity of CART cells, and reduces off-target toxicity; this strategy can be widely used in CART treatment of solid tumors such as HER2-positive breast cancer, gastric cancer, and ovarian cancer, and has significant clinical translation potential.

Description

Technical Field

[0001] The present invention relates to the field of immune cell therapy, and in particular to a CART cell and a method for preparing the same. Background Art

[0002] In recent years, immune cell therapy has made significant progress in tumor treatment, among which CART cells have attracted widespread attention due to their specific recognition of tumor antigens and strong cytotoxicity. The CART cells currently used in clinical practice mainly rely on the variable region of monoclonal antibodies to construct antigen binding domains. Although they can achieve accurate recognition of tumor cells, due to their single structure, they often have problems such as insufficient affinity, insufficient signal transduction, and poor cell proliferation and tolerance. In addition, the heterogeneity of tumor antigens and the inhibitory effects of the microenvironment also limit the therapeutic effect of traditional CART cells. In order to solve the above problems, researchers at home and abroad have tried to modify CART cells by introducing additional domains, improving linker design, and optimizing co-stimulatory signal domains, but it is still difficult to strike a balance between improving cell killing activity and persistence. Therefore, it is necessary to develop a new type of CART cell to address the shortcomings of traditional technology. Summary of the Invention

[0003] The present invention provides a HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure, and applies it to CART cell therapy.

[0004] Therefore, on one hand, the present invention discloses a HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure, wherein the specific composition of the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure from N-terminus to C-terminus is: HER2-VNAR-connector peptide-pH-responsive targeting polypeptide, wherein the amino acid sequence of HER2-VNAR is shown in SEQ ID NO.3, and the amino acid sequence of the pH-responsive targeting polypeptide is shown in SEQ ID NO.1.

[0005] Preferably, the amino acid sequence of the connecting peptide of the present invention is GGGGSGGGGSGGGGS.

[0006] On the one hand, the present invention also discloses a CART cell, which includes the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure.

[0007] On the one hand, the present invention also discloses an application of the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure in the preparation of CART cells.

[0008] HER2 is an important target for a variety of solid tumors, including breast cancer, gastric cancer, and ovarian cancer. However, traditional HER2-CART therapy faces problems such as off-target toxicity, an immunosuppressive microenvironment, and insufficient tumor infiltration. The present invention optimizes the tumor targeting of HER2-CART cells through pH-responsive regulation and the combination of high-affinity HER2 single-domain antibodies, achieving the following advantages:

[0009] (1) Reduce off-target toxicity: At neutral pH, HER2 single-domain antibodies are in a hidden state, preventing CART cells from attacking normal HER2 low-expressing tissues and improving safety.

[0010] (2) Enhanced targeting of HER2-positive tumors: In the tumor microenvironment (pH < 6.8), HER2 single-domain antibodies are exposed, accurately identifying HER2-high-expressing cells and improving the specific binding of CART cells to HER2-positive tumors.

[0011] (3) High-affinity HER2 single-domain antibodies: Compared with traditional antibodies, HER2 single-domain antibodies have a smaller molecular weight (<15kDa), stronger tumor penetration and higher stability, which can effectively enhance the killing ability of CART cells.

[0012] (4) Improve tumor infiltration ability: HER2 single-domain antibodies optimize the migration characteristics of CART cells and improve their survival rate and killing ability in the HER2-positive tumor microenvironment.

[0013] In summary, the present invention combines pH-responsive targeting peptides with HER2 single-domain antibodies to improve the targeting and anti-tumor activity of CAR T cells in HER2-positive tumors and reduce off-target toxicity. This strategy can be widely used in CAR T cells for the treatment of solid tumors such as HER2-positive breast cancer, gastric cancer, and ovarian cancer, and has significant clinical translation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 SDS-PAGE detection results of HER2-VNAR.

[0015] Figure 2 Western Blot detection results of HER2-VNAR for HER2 protein, where 1 is HER2 protein (ab190418) with a molecular weight of approximately 115kDa. DETAILED DESCRIPTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0017] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0018] Example 1: Design and preparation of pH-responsive targeting polypeptides

[0019] 1. Design of pH-responsive targeting peptides

[0020] Based on the research results, the structure was designed as: [shielding domain]-[pH-sensitive hinge]-[target binding motif].

[0021] 1. Stealth Domain: An artificially designed α-helical sequence (EVQLKE)3 is used to form a stable secondary structure at neutral pH, encapsulating the target binding motif and reducing nonspecific binding.

[0022] The advantages of this design are: hydrophilic residues (such as Ser / Thr) can be added to the α-helical region to enhance water solubility; based on the EVQLKE repeat sequence, "E" can be appropriately added to increase the overall negative charge and further stabilize the structure at neutral pH.

[0023] The designed sequence is: H-EVQLKEEVQLKEEVQLKE-L-NH2.

[0024] 2. pH-Sensitive Linker: The histidine hexamer (His6) is protonated at pH < 6.8, causing the α-helix to unfold and expose the target binding motif. Short peptide spacers (such as Gly-Gly or Ala-Ala) are added to both ends of the His segment to reduce structural rigidity and improve conformational transition efficiency. The optimized sequence is: GGHHHHHHGG.

[0025] 3. Targeting Motif: RGD variant (CRGDSPASSK) specifically binds to the tumor-associated integrin αvβ3 (CD29), enhancing targeting to tumor cells. Cys disulfide bonds improve stability but may affect affinity. C-terminal stabilizing fragments (such as Phe and Tyr) can be introduced to optimize binding. Therefore, the RGD core region can be replaced with iRGD (CRGDKGPDC) to enhance penetration. The resulting optimized sequence is: CRGDSPASSKY.

[0026] In summary, the amino acid sequence of the pH-responsive targeting polypeptide is shown in SEQ ID NO. 1, and the specific amino acid sequence is: EVQLKEEVQLKEEVQLKE-LGGHHHHHHGGCRGDSPASSKY.

[0027] 2. Construction and verification of pH-responsive targeting peptides

[0028] 1. Synthesis and folding verification of pH-responsive targeted peptides

[0029] 1.1 Peptide synthesis: Solid phase synthesis (SPPS) and Fmoc protection strategy are used and commissioned to a third-party company (such as Nanjing GenScript, etc.).

[0030] 1.2 Polypeptide Folding: The synthesized polypeptide was first dissolved in pH 7.4 PBS (containing 5% glycerol and 2 mM TCEP) to maintain the reduced state. The peptide was then dialyzed against 20 mM MES buffer at pH 6.5. The pH was gradually lowered by gradient dialysis (12 kDa dialysis bag, 4°C, 12 h) to induce conformational changes.

[0031] 1.3 Conformational Analysis (Circular Dichroism): Analyses were performed using a Jasco J-1500 at 25°C, using a 1 mm pathlength quartz cuvette; the scanning range was 190–260 nm at a scan rate of 50 nm / min. The results showed that at pH 7.4, the characteristic α-helical peaks (208 nm and 222 nm) were present, with a signal at 220 nm of -18.3 mdeg. At pH 6.5, the α-helical peaks were reduced, with the signal at 220 nm dropping to -6.7 mdeg, indicating partial conformational unfolding.

[0032] 2. Targeted Function Verification

[0033] 2.1 Surface Plasmon Resonance (SPR)

[0034] (1) Instrument: Biacore T200, sensor chip CM5;

[0035] (2) Immobilized ligand: CD29-Fc fusion protein (10 μg / mL, pH 5.0 acetate buffer) coupled to a CM5 chip, activator EDC / NHS.

[0036] (3) Experimental conditions: flow rate 30 μL / min; analysis temperature 25°C; sample solution pH 7.4 PBS & pH 6.5 MES.

[0037] (4) As shown in Table 1, the binding data show that the polypeptide of the present invention has extremely weak binding ability at pH 7.4 (RU value of only 23.5), indicating that the shielding domain effectively shields the target binding motif. At pH 6.5, the RU value increased significantly (856.7) and the KD decreased to 6.7 nM, indicating that the pH-induced conformational unfolding exposes the RGD module and enhances binding. In contrast, the traditional RGD exhibits high binding ability (KD = 8.2 nM) at pH 7.4, indicating that it is not pH-dependent and may lead to a high risk of nonspecific binding. Therefore, the binding ability of the polypeptide of the present invention is better than that of the traditional RGD at pH 6.5 (KD 6.7 vs. 8.2 nM), proving that it has better targeting.

[0038] Table 1 Surface plasmon resonance data

[0039]

[0040] Example 2: Preparation and testing of HER2 single domain antibodies

[0041] 1. Shark Immunity

[0042] 1. Select healthy sharks for immunization, using HER2 protein (ab190418) as the antigen.

[0043] 2. Immunization plan:

[0044] (1) Primary immunization: HER2 protein (100 μg) was mixed with Freund's complete adjuvant and injected subcutaneously.

[0045] (2) Booster immunization: 3 booster immunizations were performed with HER2 protein (50 μg) and Freund's incomplete adjuvant every 2 weeks.

[0046] (3) Final immunization: Blood was collected 5 days after the final immunization to extract peripheral blood lymphocytes.

[0047] 2. Shark-derived single-domain antibody gene screening

[0048] 1. RNA extraction and cDNA synthesis: Total RNA was extracted from peripheral blood lymphocytes using the TRIzol method. Reverse transcriptase was used to synthesize cDNA for use as a PCR template.

[0049] 2. Single-domain antibody gene amplification: Design specific primers to amplify the novel IgNARV gene fragment.

[0050] Forward Primer: 5'-ATGGCAGGTCAGCTGGGTAAAGG-3';

[0051] Reverse Primer: 5'-TTAGCGTAGAAGAGGACGGTGAC-3'.

[0052] (1) PCR reaction system (25 μL): cDNA 1 μL; primers (10 μM) 1 μL each; Phusion DNA polymerase 0.5 μL; dNTPs (10 mM) 0.5 μL; reaction buffer 5 μL; ddH2O to 25 μL.

[0053] (2) PCR cycle parameters: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, 35 cycles; 72°C for 10 min.

[0054] 3. Expression and Purification

[0055] (1) The HER2-VNAR gene fragment obtained by PCR amplification (as shown in SEQ ID NO. 2) was purified by agarose gel electrophoresis and then treated with NcoI and XhoI double enzyme digestion. At the same time, the pET-28a vector was digested with the same enzymes and dephosphorylated with alkaline phosphatase to prevent self-ligation. The digestion products were ligated with T4 DNA ligase and then transformed into Escherichia coli DH5α competent cells. Positive clones were selected for bacterial liquid PCR screening and sent to a sequencing company for sequencing to ensure that the VNAR gene sequence was correct.

[0056] (2) The correctly cloned recombinant pET-28a-HER2-VNAR plasmid was transformed into BL21 (DE3) competent cells and cultured in LB-kanamycin medium. When the OD600 value reached 0.6-0.8, IPTG with a final concentration of 0.5 mM was added to induce expression, and the expression was optimized at 16°C (12 h) and 37°C (4 h), respectively. After ultrasonic disruption, the His-tagged VNAR protein was purified using a Ni-NTA affinity chromatography column, and the expression and purity of the target protein were confirmed by SDS-PAGE and Western Blot. The results showed that SDS-PAGE ( Figure 1 ) showed that the molecular weight of HER2-VNAR was about 12kDa, and after purification, it was a single band with no obvious other bands. Western Blot ( Figure 2 ) confirmed that HER2-VNAR can specifically bind to HER2 protein.

[0057] 3. Binding activity and functional assays

[0058] 1. ELISA assay for HER2 binding activity: ELISA plates were coated with HER2 protein and HER2-VNAR single-domain antibodies were added at varying concentrations. OD450 values ​​were measured and Kd values ​​were calculated. The results showed that ELISA assays showed a higher affinity for HER2-VNAR (Kd = 1.2 nM) than the currently commercialized monoclonal antibody (ab237715) (Kd = 3.5 nM).

[0059] 2. Flow cytometry analysis of binding capacity: HER2-VNAR binding efficiency was analyzed by incubating the antibody with HER2-positive cells (K562 cells) and combining it with a fluorescently labeled anti-shark antibody. The results showed that the MFI (median fluorescence intensity) of HER2-VNAR binding to K562 cells was approximately 1.8-fold higher than that of the commercial monoclonal antibody (ab237715).

[0060] 3. Comparison of ADCC Activity: Using CD16+ NK cells to mediate K562 cell lysis, the ADCC activity (LDH release assay) of different antibodies (HER2-VNAR vs. a commercially available monoclonal antibody (ab237715)) was measured. The results showed that the HER2-VNAR-mediated NK cell killing rate was as high as 75.2%, higher than the 58.7% of the commercial monoclonal antibody (ab237715).

[0061] 4. CDC Activity Assay: Complement-dependent cytotoxicity (CDC) assays measure the efficiency of HER2-VNAR in complement-mediated tumor cell lysis. Results showed that HER2-VNAR achieved an 81.3% cell lysis rate in the presence of complement, significantly superior to the commercial monoclonal antibody (ab237715) (64.5%).

[0062] In summary, the HER2 shark single-domain antibody (HER2-VNAR) prepared in this invention demonstrates superior performance in affinity, cell binding, ADCC, and CDC activity compared to currently available HER2 monoclonal antibodies on the market. Its small size, high affinity, and enhanced immune-killing effects make it a promising anti-HER2 antibody candidate.

[0063] Example 3: CAR-T construction and testing

[0064] 1. CAR Structure Design

[0065] 1. Antigen binding region: The HER2-VNAR prepared in Example 2 was fused with the pH-responsive targeting polypeptide designed in Example 1, with GGGGSGGGGSGGGGS used as a connecting peptide between the two. This fusion design not only retains the specificity of the antibody, but also improves the three-dimensional conformation and affinity of the binding region, while reducing immunogenicity. The specific composition of the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure from N-terminus to C-terminus is: HER2-VNAR (SEQ ID NO. 3)-connecting peptide (GGGGSGGGGSGGGGS)-pH-responsive targeting polypeptide (SEQ ID NO. 1).

[0066] 2. Linker region: The antigen binding region is followed by the CD8α hinge and transmembrane region (CD28) to ensure structural flexibility and stability.

[0067] 3. Intracellular signaling region: connects the co-stimulatory domain and the CD3ζ signaling domain (4-1BB-CD3ζ) in series to enhance T cell activity, cytotoxicity, and tolerance.

[0068] 2. CART Cell Preparation

[0069] 1. T cell isolation and activation: peripheral blood of donors was collected and PBMCs were isolated by Ficoll-Paque density gradient centrifugation; CD3 + T cells were activated for 24 hours by adding pre-prepared anti-CD3 (1 μg / mL) and anti-CD28 (1 μg / mL) antibody magnetic beads at 37°C and 5% CO2.

[0070] 2. CAR construct preparation: The sequence of the antigen binding region was obtained by PCR amplification; this sequence was sequentially integrated into the lentiviral vector with the CD8α hinge and transmembrane region (CD28) and 4-1BB-CD3ζ through recombinant cloning technology; after Sanger sequencing to confirm the construct was correct, the lentivirus was packaged in HEK293T cells using a three-plasmid system to obtain a viral titer of ≥1×10 8 TU / mL.

[0071] 3. T cell transfection: Adjust the activated T cells to 1×10 6 Lentivirus was added to a culture medium containing polycation (Polybrene 8 μg / mL) at an MOI of 10-20 and infected by centrifugation (spinoculation, 1200 g, 90 minutes). The medium was changed 48 hours after infection to remove residual virus.

[0072] 4. Cell Expansion and Quality Control: After infection, cells were expanded in RPMI 1640 medium containing IL-2 (50-100 IU / mL) and appropriate amounts of supplemental growth factors for 7-10 days. Samples were collected every 48 hours, and CAR expression was detected by flow cytometry (labeled with anti-F(ab')2 antibody). The results showed a CAR expression rate of 91.3±3.2, a CD4 / CD8 ratio of 1:1.2, and a cell survival rate of over 95%.

[0073] 5. In vitro cytotoxicity and cytokine detection

[0074] (1) Cytotoxicity was determined by LDH release assay: at E:T = 10:1, the cell lysis rate was 80.5 ± 3.8 in 6 h;

[0075] (2) ELISA test results showed that the levels of IL-2, IFN-γ and TNF-α in the co-culture supernatant were 325±15pg / mL, 478±24pg / mL and 290±18pg / mL, respectively.

[0076] 6. Comparison experiment with other CART cells: CAR expression rate, cytotoxicity and IFN-γ of different CART cells were compared, and the results are shown in Table 2.

[0077] (1) Analysis of CAR expression rate: The CAR expression rate of the CART cells of the present invention (containing HER2-VNAR+pH-responsive targeting polypeptide) was the highest, reaching 91.3±3.2%, which was slightly higher than that of the CART cells without pH-responsive targeting polypeptide (86.2±3.1%). The CAR expression rate of traditional CAR cells was 80.0±2.8%, which was lower than that of the CART cells of the present invention, which may be related to vector construction or infection efficiency. The control T cells did not contain CAR and had an expression rate of 0. Therefore, the CART cells of the present invention (especially the version containing pH-responsive targeting polypeptide) have a higher CAR expression efficiency, indicating that the optimized construction strategy may have improved gene transduction efficiency or stability.

[0078] (2) Cytotoxicity analysis: The cytotoxicity of the CART cells of the present invention (containing pH-responsive targeting polypeptides) was the highest, reaching 80.5±3.8%, which was about 11% higher than that of the CART cells without pH-responsive targeting polypeptides (72.5±4.0%). The cytotoxicity of traditional CAR cells was 60.0±5.2%, which was significantly lower than that of the CART cells of the present invention. The cytotoxicity of the control T cells was the lowest, only 15.0±2.5%, indicating that the killing activity of non-CAR-T cells was limited. Therefore, the killing ability of the CART cells of the present invention is significantly better than that of traditional CAR cells, especially the version containing pH-responsive targeting polypeptides, which further proves the enhancing effect of this modification strategy on the anti-tumor activity of CAR-T cells.

[0079] (3) Analysis of IFN-γ secretion level: The IFN-γ release of the CART cells of the present invention (containing pH-responsive targeting polypeptides) was the highest, at 478±24pg / mL, which was significantly higher than that of the CART cells without pH-responsive targeting polypeptides (410±20pg / mL). The IFN-γ release of traditional CAR cells was 350±25pg / mL, which was significantly lower than that of the CART cells of the present invention. The IFN-γ release of control T cells was the lowest, at only 50±10pg / mL, indicating that T cells without CAR modification produced almost no specific immune response. Therefore, the IFN-γ secretion level is consistent with the cytotoxicity trend, and the CART cells of the present invention (especially the version containing pH-responsive targeting polypeptides) can more effectively activate the immune response and enhance anti-tumor activity.

[0080] (4) Summary: The CART cells of the present invention are superior to traditional CAR cells in terms of CAR expression rate, cytotoxicity and IFN-γ release, and the addition of pH-responsive targeting peptides further enhances functionality. The CART cells of the present invention containing pH-responsive targeting peptides increased cytotoxicity by approximately 34% (80.5% vs. 60.0%) and IFN-γ secretion by 37% (478 vs. 350 pg / mL) compared to traditional CAR cells, showing stronger anti-tumor potential.

[0081] The above results indicate that the optimization strategy of the CART cells of the present invention (HER2-VNAR combined with pH-responsive targeting polypeptide) may improve the targeting and anti-cancer efficacy of CAR-T cells, providing theoretical support for further clinical research.

[0082] Table 2 Data summary

[0083]

[0084] 3. In vitro killing experiment

[0085] 1. Animal Model Establishment

[0086] (1) Experimental animals: NOD / SCID mice (6-8 weeks old, either sex, weighing 18-22 g) were selected and the experiment began after 1 week of adaptive feeding. Before the experiment, the mice were housed in a sterile environment with free access to sterilized feed and ultrafiltered water.

[0087] (2) Tumor model construction: HER2-positive K562 cells were selected and cultured to the logarithmic growth phase in advance; serum-free RPMI-1640 medium was used to adjust the cell concentration to 1×10 7 100 μL (1×10 6The length (L) and width (W) of the tumor were measured every two days using a vernier caliper to calculate the tumor volume. When the tumor volume reached 0.5-1.0 cm 3 At 4 hr, the patients were randomly divided into groups and cell therapy was started.

[0088]

[0089] 2. Cell Therapy and Efficacy Evaluation

[0090] 2.1 Cell preparation and experimental grouping

[0091] (1) Experimental groups (4 groups in total, 8 mice in each group) are shown in Table 3.

[0092] Table 3 Experimental groups

[0093]

[0094] (2) Cell injection method: The cells were washed twice with PBS and adjusted to an appropriate concentration; the cells were administered by tail vein injection (100 μL slow push injection, 200 μL per mouse).

[0095] 2.2 Efficacy evaluation

[0096] (1) Tumor volume monitoring: Measure the tumor volume every 3 days and record the tumor growth curve; calculate the average tumor volume at different time points until day 21; record the tumor inhibition rate:

[0097]

[0098] (2) Survival rate monitoring: Observe and record the survival status of mice, and calculate the 21-day survival rate.

[0099] (3) Weight change monitoring: Record the weight changes of mice during the experiment and assess their overall health status.

[0100] 3. Experimental data analysis (Day 21), as shown in Table 4.

[0101] (1) CART cells of the present invention (containing HER2-VNAR+pH-responsive targeting peptide): The anti-tumor effect was the best, with an average tumor volume of 0.75 cm 3 , compared with the traditional CAR cell group (1.40cm 3 ) was significantly reduced (p<0.05); the tumor inhibition rate was as high as 75.8%, which was 21% higher than that of traditional CAR cells; the survival rate was increased to 82.5%, indicating that the treatment effect was significant; the body weight increased by 5.2%, reflecting that the mice were in good health.

[0102] (2) CART cells of the present invention (without pH-responsive targeting peptide): have good anti-tumor effect, with a tumor volume of 1.10 cm 3 , tumor inhibition rate was 64.5%; survival rate was 75%, slightly lower than that of CART cells containing pH-responsive targeting peptides; weight gain was 4.3%, showing a certain health improvement effect.

[0103] (3) Traditional CAR cells: weak anti-tumor ability, tumor volume 1.40 cm 3 The tumor inhibition rate was 54.8%; the survival rate was 62.5%, which was relatively low, indicating that the tumor killing effect was limited; the body weight increased by only 2.1%, and the health status of the animals was poor.

[0104] (4) Control group (normal saline): The tumor continued to grow and reached a volume of 3.10 cm 3 , no anti-tumor effect; the survival rate was only 25%, and most mice died; the body weight decreased by 8.5%, and the health status seriously deteriorated.

[0105] Table 4 Experimental data summary

[0106]

[0107] 4. Conclusion

[0108] The CART cells of the present invention (containing HER2-VNAR+pH-responsive targeting peptide) can significantly inhibit tumor growth, prolong survival, and improve the overall health of mice, with obvious advantages over traditional CAR-T cells. The addition of pH-responsive targeting peptides further enhances the anti-tumor activity of CAR-T cells and can optimize immune cell therapy strategies. This study demonstrates that the optimized CART cells have excellent anti-tumor effects in vivo and can be used for preclinical studies in the future.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure, characterized in that: The specific composition of the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure from N-terminus to C-terminus is: HER2-VNAR-connector peptide-pH-responsive targeting polypeptide, wherein the amino acid sequence of HER2-VNAR is shown in SEQ ID NO.3, and the amino acid sequence of the pH-responsive targeting polypeptide is shown in SEQ ID NO.

1.

2. The fusion structure according to claim 1, characterized in that: The amino acid sequence of the connecting peptide is GGGGSGGGGSGGGGS.

3. A CART cell, characterized in that The CART cell includes the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure described in claim 1.

4. An application of the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure as described in claim 1 in the preparation of CART cells.

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