Preparation method of CART cell and application of CART cell in anti-tumor treatment

Abstract

The invention discloses an HER2 (human epidermal growth factor receptor 2) single-domain antibody-pH (potential of hydrogen) response type targeting polypeptide fusion structure and application thereof in CART (chimeric antigen receptor therapy) treatment. The fusion structure combines a pH-sensitive targeting motif and a high-affinity HER2 single-domain antibody, maintains a hidden state under neutral pH, reduces non-specific binding, and has a potential potential in a tumor microenvironment (pHlt; 6.8), and the HER2 targeting motif is exposed, so that the HER2 positive tumor recognition and killing capability of the CART cells is remarkably improved, the off-target toxicity is reduced, and the curative effect is improved. Therefore, the pH response type targeting polypeptide and the HER2 single-domain antibody are combined, so that the HER2 positive tumor targeting property and the anti-tumor activity of the CART cells are improved, and the off-target toxicity is reduced; the strategy can be widely applied to CART treatment of solid tumors such as HER2 positive breast cancer, gastric cancer and ovarian cancer, and has remarkable clinical transformation potential.

Description

Technical Field

[0001] The present invention relates to the field of immunocyte therapy, and particularly to a CART cell and a preparation method thereof. Background Art

[0002] In recent years, significant progress has been made in immunocyte therapy for tumor treatment. Among them, CART cells have received extensive attention due to their specific recognition of tumor antigens and strong cytotoxicity. Currently, the CART cells used clinically mainly rely on the variable regions of monoclonal antibodies to construct antigen-binding domains. Although they can achieve precise recognition of tumor cells, due to their single structure, there are often problems such as insufficient affinity, insufficient signal transduction, and poor cell expansion and tolerance. In addition, the heterogeneity of tumor antigens and the inhibitory effect of the microenvironment also limit the therapeutic effect of traditional CART cells. 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, etc., but it is still difficult to achieve a balance between improving cell killing activity and persistence. Therefore, it is necessary to develop a new type of CART cell to solve the deficiencies in traditional technologies. 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 the one hand, the present invention discloses a HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure. The specific composition of the HER2 single-domain antibody-pH-responsive targeting polypeptide fusion structure from the N-terminus to the C-terminus is: HER2-VNAR-linker-pH-responsive targeting polypeptide, wherein the amino acid sequence of HER2-VNAR is as shown in SEQ ID NO.3, and the amino acid sequence of the pH-responsive targeting polypeptide is as shown in SEQ ID NO.1.

[0005] Preferably, the amino acid sequence of the linker in the present invention is GGGGSGGGGSGGGGS.

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

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

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

[0009] (1) Reducing off-target toxicity: At neutral pH, the HER2 single-domain antibody is in a concealed state, preventing CART cells from attacking normal tissues with low HER2 expression and improving safety.

[0010] (2) Enhancing HER2-positive tumor targeting: In the tumor microenvironment (pH < 6.8), the HER2 single-domain antibody is exposed, precisely recognizing cells with high HER2 expression and improving the specific binding of CART cells to HER2-positive tumors.

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

[0012] (4) Improving tumor infiltration ability: The HER2 single-domain antibody optimizes the migration characteristics of CART cells, increasing their survival rate and killing ability in the HER2-positive tumor microenvironment.

[0013] In summary, the present invention combines a pH-responsive targeting polypeptide with a HER2 single-domain antibody, improving the HER2-positive tumor targeting and anti-tumor activity of CART cells and reducing off-target toxicity. This strategy can be widely applied to the CART treatment of HER2-positive breast cancer, gastric cancer, ovarian cancer, and other solid tumors, with significant potential for clinical translation. 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 against HER2 protein, where 1 is HER2 protein (ab190418) with a molecular weight of approximately 115 kDa. Detailed Description of the Invention

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used 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" used herein includes any and all combinations of one or more of the related listed items.

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

[0018] Example 1: Design and Preparation of pH-Responsive Targeting Polypeptide

[0019] I. Design of pH-Responsive Targeting Polypeptide

[0020] According to the research results, the structure is designed as: [Stealth Domain]-[pH-Sensitive Linker]-[Targeting Binding Motif].

[0021] 1. Stealth Domain: An artificially designed α-helix sequence (EVQLKE)3 is adopted, which forms a stable secondary structure at neutral pH, encapsulates the targeting binding motif, and reduces non-specific binding.

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

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

[0024] 2. pH-Sensitive Linker: The hexahistidine (His6) is protonated at pH < 6.8, causing the α-helix to unfold and exposing the targeting binding motif. At the same time, short peptide spacer regions (such as Gly-Gly or Ala-Ala) are added at both ends of the His segment to reduce the structural rigidity and improve the conformational conversion efficiency. The optimized sequence is: GGHHHHHHGG.

[0025] 3. Targeting Motif: RGD variant (CRGDSPASSK), which can specifically bind to tumor-associated integrin αvβ3 (CD29), enhancing the targeting to tumor cells. Cys forms disulfide bonds to improve stability, but may affect affinity. C-terminal stabilizing fragments (such as Phe, Tyr) can be introduced to optimize the binding force. Therefore, the RGD core region can be replaced with iRGD (CRGDKGPDC) to enhance the penetration ability. The 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] II. Construction and Verification of pH-Responsive Targeting Polypeptide

[0028] 1. Synthesis and Folding Verification of pH-Responsive Targeting Polypeptide

[0029] 1.1 Polypeptide synthesis: Solid-phase synthesis (SPPS) with Fmoc protection strategy was commissioned to a third-party company (such as Nanjing Genscript, etc.) to complete.

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

[0031] 1.3 Conformational analysis (circular dichroism): Using Jasco J-1500, at a temperature of 25 °C, with a 1 mm pathlength quartz cuvette; the scanning range was 190 - 260 nm, and the scanning rate was 50 nm / min. The results showed that at pH 7.4, there were characteristic α-helix peaks (208 nm & 222 nm), and at 220 nm, it was -18.3 mdeg; at pH 6.5, the α-helix decreased, and the signal at 220 nm dropped to -6.7 mdeg, indicating partial conformational unfolding.

[0032] 2. Targeting 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) was coupled to the CM5 chip with the activator EDC / NHS.

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

[0037] (4) The binding data are shown in Table 1. The binding ability of the polypeptide of the present invention is extremely weak at pH 7.4 (RU value is only 23.5), indicating that the shielding domain effectively shields the targeting binding motif. At pH 6.5, the RU value increases significantly (856.7), and the KD decreases to 6.7 nM, indicating that the pH-induced conformational unfolding exposes the RGD module and enhances the binding. While the traditional RGD shows high binding force at pH 7.4 (KD = 8.2 nM), indicating that it has no pH-dependence and may lead to a high risk of non-specific binding. Therefore, the polypeptide of the present invention has better binding force than the traditional RGD at pH 6.5 (KD 6.7 vs. 8.2 nM), proving its better targeting property.

[0038] Table 1 Surface plasmon resonance data

[0039]

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

[0041] I. Shark immunization

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

[0043] 2. Immunization protocol:

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

[0045] (2) Booster immunization: Every two weeks, use HER2 protein (50 μg) and Freund's incomplete adjuvant for three booster immunizations.

[0046] (3) Final immunization: Blood is taken 5 days after the final immunization, and peripheral blood lymphocytes are extracted.

[0047] II. Screening of shark-derived single-domain antibody genes

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

[0049] 2. Amplification of single-domain antibody genes: Specific primers are designed to amplify the novel IgNAR V gene fragment.

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

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

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

[0053] (2) PCR cycling 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, digested with NcoI and XhoI double enzymes, and at the same time, the pET-28a vector was digested with the same enzymes and dephosphorylated with alkaline phosphatase to prevent self-ligation. The digested products were ligated by T4 DNA ligase and then transformed into Escherichia coli DH5α competent cells. Positive clones were picked for colony 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 using LB-kanamycin medium. When the OD600 value reached 0.6 - 0.8, IPTG with a final concentration of 0.5 mM was added for induction of expression, and the expression was optimized under the conditions of 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 approximately 12 kDa, a single band after purification, and no obvious impurity bands. Western Blot ( Figure 2 ) confirmed that HER2-VNAR could specifically bind to HER2 protein.

[0057] III. Detection of binding activity and function

[0058] 1. ELISA detection of HER2 binding activity: Coat an ELISA plate with HER2 protein, add HER2-VNAR single-domain antibodies at different concentrations, detect the OD450 value, and calculate the Kd value. The results show that, as determined by ELISA, HER2-VNAR (Kd = 1.2 nM) has a higher affinity than the commercially available monoclonal antibody (ab237715) (Kd = 3.5 nM).

[0059] 2. Flow cytometry detection of binding ability: Incubate the antibody with HER2-positive cells (K562 cells), bind the fluorescently labeled anti-shark antibody, and analyze the binding efficiency of HER2-VNAR. The results show that the binding MFI (median fluorescence intensity) of HER2-VNAR to K562 cells is approximately 1.8 times higher than that of the commercially available monoclonal antibody (ab237715).

[0060] 3. Comparison of ADCC activity: Use CD16+ NK cells to mediate the lysis of K562 cells, and measure the ADCC activity (LDH release method) of different antibodies (HER2-VNAR vs. commercially available monoclonal antibody (ab237715)). The results show that the killing rate of NK cells mediated by HER2-VNAR is as high as 75.2%, which is higher than 58.7% of the commercially available monoclonal antibody (ab237715).

[0061] 4. CDC activity detection: Complement-dependent cytotoxicity (CDC) assay to measure the efficiency of HER2-VNAR in complement-mediated tumor cell lysis. The results show that the cell lysis rate of HER2-VNAR in the presence of complement reaches 81.3%, which is significantly better than that of the commercially available monoclonal antibody (ab237715) (64.5%).

[0062] In summary, compared with the existing HER2 monoclonal antibodies on the market, the HER2 shark single-domain antibody (HER2-VNAR) prepared in the present invention exhibits superior performance in terms of affinity, cell binding ability, ADCC, and CDC activities. Its small size, high affinity, and more potent immune killing effect make it a more promising anti-HER2 candidate antibody.

[0063] Example 3: Construction and testing of CAR-T

[0064] I. CAR structure design

[0065] 1. Antigen-binding region: HER2-VNAR prepared in Example 2 was used, and then fused with the pH-responsive targeting polypeptide designed in Example 1. GGGGSGGGGSGGGGS was used as the linker peptide between the two. This fusion design not only retains the specificity of the antibody, but also improves the conformational 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 the N-terminus to the C-terminus is: HER2-VNAR (SEQ ID NO.3)-linker peptide (GGGGSGGGGSGGGGS)-pH-responsive targeting polypeptide (SEQ ID NO.1).

[0066] 2. Linker region: The CD8α hinge and transmembrane region (CD28) were sequentially connected after the antigen-binding region to ensure structural flexibility and stability.

[0067] 3. Intracellular signaling region: The co-stimulatory domain and CD3ζ signal transduction domain (4-1BB-CD3ζ) were tandemly connected to enhance the activity, cytotoxicity and tolerance of T cells.

[0068] II. Preparation of CAR-T cells

[0069] 1. T cell isolation and activation: Peripheral blood of the donor was collected, and PBMCs were separated by Ficoll-Paque density gradient centrifugation; CD3 + T cells were purified by magnetic bead method, and pre-prepared anti-CD3 (1 μg / mL) and anti-CD28 (1 μg / mL) antibody magnetic beads were added, and activated at 37 °C and 5% CO2 for 24 hours.

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

[0071] 3. T cell transfection: The activated T cells were adjusted to 1×10 6 cells / mL, and lentivirus was added under the condition of MOI = 10 - 20 in the culture medium containing polycation (Polybrene 8 μg / mL), and centrifuged for infection (spinoculation, 1200 g, 90 minutes). The culture medium was changed 48 hours after infection to remove residual virus.

[0072] 4. Cell expansion and quality control: After infection, expand for 7 - 10 days in RPMI 1640 medium containing IL-2 (50 - 100 IU / mL) and appropriate supplemented growth factors; collect samples every 48 hours and detect CAR expression by flow cytometry (labeled with anti-F(ab')2 antibody). The results showed that the CAR expression rate was 91.3 ± 3.2, the CD4 / CD8 ratio was 1:1.2, and the cell survival rate was over 95%.

[0073] 5. In vitro cytotoxicity and cytokine detection

[0074] (1) The cytotoxicity was determined by the LDH release method: When E:T = 10:1, the cell lysis rate at 6 hours was 80.5 ± 3.8;

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

[0076] 6. Comparative experiments with other CART cells: By comparing the CAR expression rate, cytotoxicity, and IFN-γ of different CART cells, the results are shown in Table 2.

[0077] (1) CAR expression rate analysis: 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%, 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%, 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 the expression rate was 0. Therefore, the CART cells of the present invention (especially the version containing pH-responsive targeting polypeptide) have higher CAR expression efficiency, indicating that the optimized construction strategy may improve gene transduction efficiency or stability.

[0078] (2) Cytotoxicity analysis: The cytotoxicity of the CART cells of the present invention (containing pH-responsive targeting polypeptide) was the highest, reaching 80.5 ± 3.8%, which was about 11% higher than that of the CART cells without pH-responsive targeting polypeptide (72.5 ± 4.0%). The cytotoxicity of traditional CAR cells was 60.0 ± 5.2%, 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 is 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 polypeptide, further demonstrating the enhancing effect of this modification strategy on the anti-tumor activity of CAR-T cells.

[0079] (3) IFN-γ secretion level analysis: The IFN-γ release of the CART cells (containing pH-responsive targeting polypeptide) of the present invention is the highest, which is 478 ± 24 pg / mL, significantly higher than that of the CART cells without pH-responsive targeting polypeptide (410 ± 20 pg / mL). The IFN-γ release of traditional CAR cells is 350 ± 25 pg / mL, which is significantly lower than that of the CART cells of the present invention. The IFN-γ release of control T cells is the lowest, only 50 ± 10 pg / mL, indicating that T cells without CAR modification hardly produce specific immune responses. Therefore, the IFN-γ secretion level is consistent with the cytotoxicity trend. The CART cells of the present invention (especially the version containing pH-responsive targeting polypeptide) can more effectively activate the immune response and enhance the 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 polypeptide further enhances the functionality. The CART cells of the present invention containing pH-responsive targeting polypeptide have about 34% higher cytotoxicity (80.5% vs. 60.0%) and 37% higher IFN-γ secretion (478 vs. 350 pg / mL) than 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] Data summary in Table 2

[0083]

[0084] III. In vitro killing experiment

[0085] 1. Establishment of animal model

[0086] (1) Experimental animals: NOD / SCID mice (6 - 8 weeks old, both male and female, weighing 18 - 22 g) were selected and started the experiment after 1 week of adaptive feeding; before the experiment, the mice were all raised in a sterile environment and freely obtained sterilized feed and ultrafiltered water.

[0087] (2) Construction of tumor model: HER2-positive K562 cells were selected and pre-cultured to the logarithmic growth phase; the cell concentration was adjusted to 1×10 7 cells / mL using serum-free RPMI-1640 medium; 100 μL (1×10 6A cell suspension; The length (L) and width (W) of the tumor were measured every two days using a vernier caliper, and the tumor volume was calculated. When the tumor volume reached 0.5 - 1.0 cm 3 , randomly grouped, and cell therapy was started.

[0088]

[0089] 2. Cell Therapy and Efficacy Evaluation

[0090] 2.1 Cell Preparation and Experimental Grouping

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

[0092] Table 3 Experimental Grouping

[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 (slowly injected at 100 μL, 200 μL for each mouse).

[0095] 2.2 Efficacy Evaluation

[0096] (1) Tumor volume monitoring: The tumor volume was measured every 3 days, and the tumor growth curve was recorded; 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 the mice, and count the survival rate at 21 days.

[0099] (3) Body weight change monitoring: Record the body weight change of the mice during the experiment and evaluate the overall health status.

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

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

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

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

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

[0105] Table 4 Summary of experimental data

[0106]

[0107] 4. Conclusions

[0108] The CART cells of the present invention (containing HER2-VNAR + pH-responsive targeting polypeptide) can significantly inhibit tumor growth, prolong the survival period, and improve the overall health status of mice, showing obvious advantages over traditional CAR-T cells. The addition of the pH-responsive targeting polypeptide further enhances the anti-tumor activity of CAR-T cells and can optimize the immunocyte therapy strategy. This study proves 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 embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope 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-connecting 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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