Immune cells targeting fshr and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to an immune cell targeting FSHR and application thereof. The amino acid sequence of follicle stimulating hormone is used as an extracellular antigen binding region of a chimeric antigen receptor, which can specifically recognize and target target cells related to follicle stimulating hormone disorders, exhibit stronger cell killing ability, and effectively prevent immune rejection reaction caused by the chimeric receptor. Therefore, the technical scheme provided by the present application can be used for drug development, and then related therapeutic drugs with significant anti-tumor and immunoregulatory effects and lower side effects can be obtained.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an immune cell that targets FSHR and its applications. Background Technology

[0002] Cancer is a leading cause of death worldwide and severely limits the extension of human life expectancy. Malignant tumors related to women, including breast cancer, ovarian cancer, and endometrial cancer, seriously affect women's physical and mental health. Ovarian cancer, as one of the most common gynecological malignancies, has a high incidence rate. Furthermore, due to the lack of specific early symptoms and effective early screening methods, as well as the dominance of aggressive high-grade serous ovarian cancer in ovarian cancer cases, the prognosis of ovarian cancer is poor, and the five-year survival rate is generally low.

[0003] Currently, most ovarian cancer patients are treated with a comprehensive approach combining surgery and chemotherapy. Surgery plays a crucial role in initial treatment, while chemotherapy is typically used as adjuvant therapy to reduce micrometastases and potential metastases. Although surgery can effectively remove visible tumors and alleviate symptoms to some extent, early-stage ovarian cancer often presents with no obvious symptoms and is prone to micrometastases. Therefore, postoperative chemotherapy is still necessary to reduce the risk of recurrence and improve survival prognosis. Chemotherapy can effectively reduce the risk of local tumor recurrence and prolong overall survival to some extent, but its side effects, such as nausea, vomiting, hair loss, and decreased immune function, significantly impact patients' quality of life and may lead to long-term health problems. Therefore, new treatment strategies need to be explored to improve treatment efficacy and reduce side effects.

[0004] Among these, chimeric antigen receptor (CAR) immunotherapy, an important research direction in the field of tumor treatment, is considered to offer a promising new solution for ovarian cancer treatment. A chimeric antigen receptor is an artificially designed fusion protein, mainly composed of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular activation domain. The extracellular antigen-binding domain typically contains two distinct single-chain variable regions for specifically recognizing antigens on the surface of tumor cells. When the chimeric antigen receptor recognizes and binds to tumor antigens, its intracellular activation domain is activated, thereby inducing immune cells to release perforin, granzymes, and cytokines to directly kill tumor cells, thus achieving an anti-tumor therapeutic effect.

[0005] Although immunotherapy based on chimeric antigen receptors has shown great potential in the treatment of solid tumors and has achieved initial success in some malignant tumors, further innovation and optimization in drug development are needed to effectively apply it to the treatment of ovarian cancer. Summary of the Invention

[0006] This invention uses the amino acid sequence of follicle-stimulating hormone (FSHR) as the extracellular antigen-binding region of a chimeric antigen receptor to provide an immune cell that targets FSHR and its application.

[0007] In a first aspect, the present invention provides an engineered immune cell that expresses a chimeric antigen receptor; the chimeric antigen receptor includes an FSHR binding domain; the immune cell is selected from any one or more of γδT cells, NKT cells, natural killer cells, B cells, macrophages, and DC cells.

[0008] In some embodiments, the FSHR binding domain comprises or is derived from a single-chain antibody comprising FSHβ of the amino acid sequence shown in SEQ ID NO:1 and FSHα of the amino acid sequence shown in SEQ ID NO:2.

[0009] In some embodiments, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, the FSHR binding domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain.

[0010] In some embodiments, the intracellular signal transduction domains are derived from: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d.

[0011] In some embodiments, the intracellular signal transduction domain further comprises an intracellular co-stimulatory sequence.

[0012] In some embodiments, the intracellular co-stimulatory sequence is derived from any one or more of the following: CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, and CD83 ligands.

[0013] In some embodiments, the single-chain antibody in the engineered immune cells described above is replaced with a single-domain antibody.

[0014] Secondly, the present invention provides an isolated nucleic acid molecule that encodes the chimeric antigen receptor in an engineered immune cell as described in any of the preceding claims.

[0015] Thirdly, the present invention provides an expression vector comprising the above-described isolated nucleic acid molecules.

[0016] In some embodiments, the expression vector is selected from any one or more of lentiviral vectors, retroviral vectors, adenoviral vectors, transposons, DNA, RNA, and plasmids.

[0017] Fourthly, the present invention provides a method for preparing engineered immune cells, comprising: transfecting or transducing immune cells using the above-mentioned expression vector to obtain the engineered immune cells; wherein the immune cells are selected from any one or more of γδT cells, NKT cells, natural killer cells, B cells, macrophages and DC cells.

[0018] Fifthly, the present invention provides a pharmaceutical composition comprising the engineered immune cells described in any one of the preceding claims, the isolated nucleic acid molecules described above, the expression vector described above, or the engineered immune cells prepared by the method described above for preparing engineered immune cells, and a physiologically acceptable excipient.

[0019] In a sixth aspect, the present invention provides the use of any of the above-described engineered immune cells, the above-described isolated nucleic acid molecules, the above-described expression vector, or engineered immune cells prepared by the above-described method for preparing engineered immune cells, or the above-described pharmaceutical composition in the preparation of a medicament for treating follicle-stimulating hormone-related diseases.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention uses the amino acid sequence of follicle-stimulating hormone (FSH) as the extracellular antigen-binding region of a chimeric antigen receptor, providing a chimeric antigen receptor, binding protein, isolated nucleic acid molecule, expression vector, and engineered immune cells that target the follicle-stimulating hormone receptor (FSHR).

[0022] These constructs significantly upregulate the expression of FSH-related RNA and proteins within cells, specifically recognize and target target cells associated with FSH symptoms, especially ovarian cancer cells, and exhibit enhanced cell-killing capabilities. Furthermore, because the target is a chimeric endocrine receptor with a binding domain derived from a natural ligand, maximum specificity is ensured while effectively preventing immune rejection responses induced by the chimeric receptor. Moreover, the technical solution provided by this invention has broad application prospects and can be used in drug development to obtain therapeutic drugs with significant anti-tumor and immunomodulatory effects and low side effects. Attached Figure Description

[0023] Figure 1This is a diagram of the FSH CAR recombination structure in one embodiment;

[0024] Figure 2 This is a structural diagram of an FSH CAR transfer plasmid in one embodiment;

[0025] Figure 3A , 3B This is the qPCR validation result for FSHα and FSHβ in one embodiment. Figure 3C This is a Western Blot verification result from one embodiment;

[0026] Figure 4A , 4B 4C and 4D are titer measurement results in one embodiment, wherein... Figure 4A The results are for the blank control. Figures 4B to 4D Results for 1 μL, 5 μL, and 10 μL of FSH CAR lentivirus, respectively;

[0027] Figure 5A , 5B Figure 5C shows the flow cytometry detection results in one embodiment, where... Figure 5A The results are for NK92 cells that have not been transduced with CAR. Figure 5B This is the result of treating cells with a non-specific antibody of the same isotype as the detection antibody. Figure 5C Results for NK92 cells transduced with FSH CAR;

[0028] Figure 6 This is a diagram showing the expression of FSHR in different cell types in one embodiment;

[0029] Figure 7A , 7B 7C, 7D, and 7E are diagrams illustrating the killing effect of immune cells on target cells in one embodiment. Figures 7A to 7E The target cells were CAOV3, A2780, OVCAR3, NIC-H727, and U87, respectively. Detailed Implementation

[0030] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] Example 1: Construction of a chimeric antigen receptor targeting FSHR

[0032] (1) Structural design of FSH CAR.

[0033] Human FSHβ (SEQ ID NO:1) and CGα (SEQ ID NO:2) peptides were selected and linked together using a linker containing glycine and serine (SEQ ID NO:3) to form a peptide fusion. CGα is the common α subunit of gonadotropins such as FSH, LH, hCG, and TSH, and its structure is identical to FSHα. Subsequently, this fusion peptide was linked to the hinge region and transmembrane domains (i.e., CD8H and CD8TM) of human CD8α, and further bound to intracellular signal transduction fragments of 4-1BB and CD3ζ. Finally, a [synthesis / design] was created. Figure 1 The FSH CAR recombination structure shown forms the FSHβ-FSHα-CD8H-CD8TM-4-1B B-CD3ζ molecule.

[0034] Table 1. Sequence listing of some expression elements in FSH CAR

[0035]

[0036]

[0037] (2) Construction of FSH CAR transfer plasmid.

[0038] After codon optimization of the sequence obtained in step (1), DNA sequence synthesis and PCR amplification were performed. The amplified DNA fragment was then cloned into an empty transfer plasmid pRLenti-EF1a-MCS-3xFLAG-WPRE containing an ampicillin resistance marker. This vector contains a multiple cloning site, facilitating subsequent gene construction and manipulation. Subsequently, the constructed plasmid was transformed into competent *E. coli* cells, and positive monoclonal strains were obtained through resistance selection. The strains were then amplified and cultured, and the plasmid was extracted.

[0039] Ultimately, the following was constructed Figure 2 The FSH CAR transfer plasmid structure shown includes, in sequence, the EF1a promoter, FSHβ and FSHα gene sequences, and the WPRE sequence. Furthermore, the extracted plasmid was sequenced, and after confirming the sequence was correct, the bacterial strain was amplified and cultured, and the plasmid was extracted for subsequent steps.

[0040] Example 2: Validation of FSH CAR transfer plasmid

[0041] (1) Validation of FSH CAR transfer plasmid.

[0042] 293T cells in good growth condition were collected. When the cell density reached 80%, the cell culture medium was replaced with Opti-MEM medium. Then, the FSH CAR transfer plasmid obtained in step (2) of Example 1 was used, along with Lipofectamine.TM Cells were transfected with 3000 transfection reagent to obtain cells expressing FSH CAR.

[0043] Specifically, follow the instructions to dilute Lipofectamine with Opti-MEM medium. TM 3000, and P3000 was added to the Opti-MEM medium at the same time. TM The reagent is used to dilute the DNA. The diluted DNA is then mixed with Lipofectamine. TM Mix reagent 3000 at a 1:1 ratio and incubate at room temperature for 10-15 minutes. Then, add the mixture to 6-well plates containing seeded cells and incubate at 37°C with 5% CO2 for 6 hours. After transfection, aspirate the transfection reagent and replace with fresh culture medium.

[0044] (2) q-PCR verification.

[0045] 48 h after transfection, total RNA was extracted from the cells expressing FSH CAR obtained in step (1), cDNA was synthesized using reverse transcriptase, and the expression level of FSH gene was detected by qPCR, with GAPDH as the internal reference gene.

[0046] Specifically, 293T cells expressing FSH CAR obtained in step (1) were digested with trypsin to prepare a cell suspension. After washing once with PBS, 2-3 mL of RNA-easy lysis buffer was added to the cell suspension, 500 μL per well for a 6-well plate, to fully lyse the cells. Then, 2 / 5 volume of RNase-free ddH2O was added to the lysis buffer, inverted to mix, and allowed to stand for 5 min. Subsequently, the cells were centrifuged at 12000g for 15 min, the upper aqueous phase was collected and mixed with an equal volume of isopropanol, and allowed to stand for 10 min. After that, the supernatant was removed by centrifugation, 500 μL of 75% ethanol was added, inverted to mix, and the supernatant was removed by centrifugation again. Finally, the precipitate was air-dried at room temperature, and an appropriate amount of RNase-free ddH2O was added to completely dissolve the RNA precipitate.

[0047] (3) Western Blot verification.

[0048] 293T cells expressing FSH CAR obtained in step (1) were lysed using RIPA lysis buffer to extract total cellular protein. The concentration of extracted protein samples was determined using a BCA protein quantification kit, and the loading volume per well was adjusted to 30 μg based on the results. Subsequently, proteins were separated by SDS-PAGE electrophoresis under the following conditions: pre-electrophoresis at 80V for 30 min, followed by electrophoresis at 120V for 60 min to separate proteins of different molecular weights. After protein separation, the proteins were transferred to a PVDF membrane and transferred at 95V for 90 min to complete the transfer. After transfer, the PVDF membrane was placed in 5% BSA blocking buffer and blocked at room temperature for 1 h. Then, primary antibody targeting FSH was added at a 1:500 dilution, and the membrane was incubated overnight at 4°C. After incubation, the membrane was washed with Tris-HCl buffer containing Tween 20 to remove non-specific binding. Next, diluted secondary antibody was added, and the membrane was incubated at room temperature for 1 h. Finally, the protein signal was detected and visualized using an ECL chemiluminescence detection system.

[0049] result: Figure 3A and Figure 3B The qPCR validation results for FSHα and FSHβ are shown separately. It can be seen that, compared with the control group, the mRNA levels of FSHα and FSHβ were higher in 293T cells transfected with the FSH CAR transfer plasmid. Figure 3C The results of Western blotting are shown. It can be seen that, compared with the control group, the expression level of FSH protein was higher in 293T cells transfected with the FSH CAR transfer plasmid. Therefore, transfection with the FSH CAR transfer plasmid can significantly upregulate the expression of intracellular FSH-related RNA and FSH protein.

[0050] Example 3: Preparation of FSH CAR Lentiviral Virus

[0051] (1) Packaging of FSH CAR lentivirus.

[0052] A three-plasmid transfection system was established using the packaging plasmid (psPAX2), the envelope plasmid (pMD2.G), and the FSH CAR transfer plasmid obtained in step (2) of Example 1. HEK 293T cells were co-transfected to prepare lentiviral particles.

[0053] Specifically, HEK 293T cells were passaged within 24 hours before transfection to ensure that the cells used were healthy cells that had been passaged no more than 10 times, and then seeded in 10cm cell culture dishes. At the time of plasmid transfection, the cells needed to be in good growth condition, fully adherent, and have a cell density of approximately 80%.

[0054] The transfer plasmid, packaging plasmid, and envelope plasmid were dissolved in 0.5 mL of Opti-MEM medium at a ratio of 5 μg:3 μg:2 μg and mixed well. Simultaneously, 25 μL of liposomes and 0.5 mL of Opti-MEM medium were added to another centrifuge tube and mixed well. Then, the solution containing the plasmids and the solution containing the liposomes were mixed at a 1:1 volume ratio and incubated at room temperature for 20 min to form the transfection complex.

[0055] The transfection complex was slowly added to the 293T cell culture dish to be transfected, and gently shaken to mix thoroughly. The cell culture medium was DMEM complete medium without penicillin and streptomycin. The cells were then incubated at 37°C in a 5% CO2 incubator for 6–8 hours. Afterward, the medium was replaced with fresh medium and cultured for another 48 hours. The culture supernatant was collected, fresh medium was added, and the cells were cultured for another 72 hours. The culture supernatant was collected again for subsequent experiments.

[0056] (2) Concentration of FSH CAR lentivirus.

[0057] The viral supernatants collected 48h and 72h after transfection in step (1) were mixed, centrifuged at 4℃ and 3500g for 10min to remove cell debris, and the supernatant was filtered through a 0.45μm filter membrane.

[0058] The viral supernatant was concentrated using a universal viral concentration kit. Specifically, the viral supernatant was mixed with lentivirus concentration reagent at a volume ratio of 3:1 and incubated on a shaker at 4°C for 8 hours. Afterward, it was centrifuged at 4°C and 3500g for 40 minutes, the supernatant was discarded, and the virus-containing precipitate was collected.

[0059] Based on the original supernatant volume, add 1 / 10 volume of virus resuspension to the precipitate, incubate at 4°C for 10 min, and then gently resuspend and reconstitute the virus precipitate by pipetting. Finally, centrifuge the resuspension at 4°C and 12000g, collect the virus-containing supernatant, aliquot it, and store it at -80°C for subsequent experiments.

[0060] (3) Determination of FSH CAR lentivirus titer.

[0061] Prepare cell suspensions from well-grown HEK 293T cells at a ratio of 3 × 10⁻⁶. 5 Cells were seeded at a uniform density in 24-well cell culture plates, and polybrene, an infection-promoting agent, was added to each well of the culture medium at a final concentration of 5 μg / mL to enhance infection efficiency.

[0062] Next, the virus-containing supernatant obtained in step (2) was added to the 24-well plate, gently patted to mix, and then incubated in an incubator for 48 hours. The volume of the virus-containing supernatant was 1, 5, or 10 μL. As a control, one well without virus was also included.

[0063] Forty-eight hours after infection, cells were collected and digested with trypsin to prepare a single-cell suspension. The positivity rate of infected cells (the percentage of positive cells out of the total cells) was then determined by flow cytometry, and the lentivirus titer was calculated using the following formula: Lentiviral titer (TU / mL) = 3 × 10⁻⁶. 5 × Positive rate / Lentiviral volume (mL).

[0064] result: Figure 4A , 4B 4C and 4D show the titer determination results, among which Figure 4A The results are for the blank control. Figures 4B to 4D Results for 1 μL, 5 μL, and 10 μL of FSH CAR lentivirus, respectively.

[0065] The results showed that the titer of FSH CAR lentivirus was 1.8 × 10⁻⁶. 7 TU / mL, meaning 1.8 × 10⁻⁶ FSH CAR lentiviral particles with transduction capability per milliliter of viral suspension. 7 indivual.

[0066] Example 4: Preparation of FSH CAR-NK92 cells

[0067] NK92 cells were infected with the virus-containing supernatant obtained in step (2) of Example 3 at an MOI ratio of 20. During lentiviral infection, the final concentration of IL-2 in the culture medium was adjusted to 400 IU / mL, and the concentration of polybrene was adjusted to 7.5 μg / mL to improve infection efficiency. Five hours after infection, fresh culture medium containing 200 IU / mL of IL-2 was added, and the cells were cultured for another 48 hours. The successfully infected NK92 cells were then identified as the target cells, FSH CAR-NK92 cells. Subsequently, the expression of FSH in the infected NK92 cells was analyzed by flow cytometry to assess the positive rate of viral infection and verify the infection effect.

[0068] result: Figure 5A , 5B 5C shows the flow cytometry results, in which Figure 5A The results are for NK92 cells that have not been transduced with CAR. Figure 5B This is the result of treating cells with a non-specific antibody of the same isotype as the detection antibody. Figure 5C Results for NK92 cells transduced with FSH CAR.

[0069] The results showed that the positive rate of FSH CAR lentiviral infection was 63.6%, with high transduction efficiency. Meanwhile, compared with... Figure 5A and 5B compared to, Figure 5C The presence of a clearly right-shifted positive signal peak indicates that FSH CAR is stably expressed in NK92 cells and located on the NK92 cell membrane, meaning that the NK92 cell genome contains expression cassettes of the FSHα and FSHβ chains. Furthermore, the high proportion of FSH CAR-positive cells further suggests that it can more effectively exert its anti-tumor and immunomodulatory effects.

[0070] Example 5: Identification of target cells

[0071] Different cell lines were selected for culture, including FSHR-positive ovarian cancer cell lines OV CAR3, CAOV3, and A2780, and FSHR-negative lung cancer cell lines NIC-H727 and glioma cell line U87. Total RNA was then extracted from these cells using an RNA extraction kit, and reverse transcription was performed to synthesize cDNA. The expression level of FSHR in different cell lines was analyzed based on RT-PCR.

[0072] result: Figure 6 The expression of FSHR in different cell types was shown. It can be seen that FSHR expression differs among cell types, with it mainly expressed in ovarian cancer cells A2780, OVCAR3, and CAOV3, while it was not detected in lung cancer cells NIC-H727 and glioma cells U87.

[0073] Example 6: Evaluation of the killing ability of FSH CAR-NK92 cells

[0074] Cell densities of OVCAR3, CAOV3, A2780, NIC-H727, and U87 cell lines were adjusted, single-cell suspensions were prepared, and seeded into 6-well plates. Subsequently, luciferase lentivirus was added, and the medium was replaced with fresh medium after 24 hours. Antibiotics were added at 48 hours for selection, and the selection process lasted for 14 days.

[0075] Cell counts were performed on OVCAR3, CAOV3, A2780, NIC-H727, and U87 cell lines that were in good growth condition and contained luciferase, and the cell density was adjusted to 1×10⁻⁶. 4 Cells / well were seeded onto low-adsorption 96-well plates.

[0076] FSH CAR-NK92 cells obtained in Example 4 were seeded into the target cells at effector-to-target ratios of 1:1 and 4:1, with three replicates and a final volume of 200 μL per well. After mixing, the culture plates were incubated at 37°C in a 5% CO2 incubator for 16 h. After incubation, the killing efficiency of the cells was assessed using a firefly luciferase reporter gene assay kit. As a control, NK92 cells were used in the same manner.

[0077] result: Figure 7A , 7B 7C, 7D, and 7E demonstrate the cytotoxic effect of immune cells on target cells, among which... Figures 7A to 7E The target cells were CAOV3, A2780, OVCAR3, NIC-H727, and U87, respectively.

[0078] It can be seen that, at the same effector-to-target ratio, FSH CAR-NK92 cells showed a higher killing effect on CAOV3, A2780, and OVCA R3 than NK92 cells, with the killing effect being more than 2-fold enhanced, indicating that FSH CAR-NK92 cells have a stronger killing effect on ovarian cancer. Furthermore, in the FSHR-negative lung cancer cell line NIC-H727 and the glioma cell line U87, FSH CAR-NK92 cells did not show an enhanced killing effect, indicating that it has effective targeting.

[0079] Example 7: Application of chimeric antigen receptors targeting FSHR

[0080] This embodiment provides the application of a chimeric antigen receptor targeting FSHR, and the isolated nucleic acid molecules, expression vectors, and engineered immune cells obtained based on the chimeric antigen receptor in the preparation of drugs for treating FSH-related diseases.

[0081] The method for preparing this drug includes at least the following steps: mixing the aforementioned chimeric antigen receptor targeting FSHR, binding protein, isolated nucleic acid molecule, expression vector, or engineered immune cells with a physiologically acceptable excipient to obtain a drug for treating FSH-related conditions. The engineered immune cells are obtained by transfecting or transducing any one or more of the following immune cell types using the expression vector: γδT cells, NKT cells, natural killer cells, B cells, macrophages, and dendritic cells.

[0082] The resulting drug can specifically identify and target target cells associated with FSH, especially ovarian cancer cells, and exhibits stronger cell-killing capabilities. Furthermore, because the target is a chimeric endocrine receptor with a binding domain derived from a natural ligand, it effectively prevents chimeric receptor-induced immune rejection while ensuring maximum specificity. Therefore, this drug has significant anti-tumor and immunomodulatory effects with low side effects, and shows broad application prospects.

[0083] What needs to be understood is:

[0084] (1) The above embodiments use 4-1BB and CD3ζ as intracellular signal transduction domains to design chimeric antigen receptors targeting FSHR, but the technical solution provided by the present invention has wide applicability. In practical applications, other intracellular signal transduction domains such as FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d can be used, or any one or more of the following ligands can be used as intracellular co-stimulatory sequences: CD27, CD28, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, and CD83. Alternatively, no intracellular co-stimulatory sequence may be used. This should not be construed as a limitation of this application.

[0085] (2) The above embodiments use pRLenti-EF1a-MCS-3xFLAG-WPRE transfer plasmid and lentivirus as expression vectors, but the technical solution provided by this invention has wide applicability. In practical applications, other gene transfer systems such as retroviral vectors, adenovirus vectors, transposons, DNA, RNA and plasmids can also be used, which should not be construed as a limitation of this application.

[0086] (3) The above embodiments use HEK 293T cells as host cells to construct gene expression systems, but the technical solutions provided by this invention have wide applicability. In practical applications, host cells such as CHO cells and Vero cells can also be used to construct gene expression systems, which should not be construed as a limitation of this application.

[0087] (4) In the above embodiments, NK92 cells were engineered as immune cells to obtain FSH CAR-NK92 cells, which express chimeric antigen receptors or binding proteins targeting FSHR, exhibiting a broader spectrum of anti-tumor effects. This significantly reduces the risk of immune rejection and the probability of cytokine storms, and weakens the influence of PD-1 inhibition in the tumor microenvironment. However, the technical solution provided by this invention has broad applicability. In practical applications, any one or more immune cells, including γδT cells, NKT cells, natural killer cells, B cells, macrophages, and DC cells, can also be engineered; this should not be construed as a limitation of this application.

[0088] (5) The above embodiments use ovarian cancer cell lines OVCAR3, CAOV3, and A2780 as target cells and describe specific implementation methods of cells modified with chimeric antigen receptors to target FSHR. However, the technical solution provided by this invention has broad applicability in terms of treatment mechanism. In practical applications, it can also be applied to other follicle-stimulating hormone-related diseases such as premature ovarian failure, ovarian insufficiency, pituitary adenoma, and polycystic ovary syndrome. This should not be construed as a limitation of this application.

[0089] Furthermore, based on the technical solutions provided by this invention, other anti-tumor treatment methods such as anti-PD-1 antibody therapy and CAR-T therapy can be combined, or treatment and drug development can be carried out in combination with surgery, chemotherapy, radiotherapy, ablation, intervention, etc., which should not be construed as a limitation of this application.

[0090] Related terms:

[0091] Follicle-stimulating hormone (FSH) is a 35.5 kDa diglycoprotein composed of two polypeptide subunits: an α chain and a β chain. The α chain consists of 92 amino acid residues, identical to the α chains of luteinizing hormone, thyroid-stimulating hormone, and human chorionic gonadotropin (hCG). The β chain consists of 111 amino acid residues and is responsible for the specific binding and interaction with the FSH receptor.

[0092] The follicle-stimulating hormone receptor (FSHR) is a membrane protein specifically expressed in the ovary and testis, and its expression is significantly increased in ovarian cancer tissue. Its structure includes an N-terminal extracellular region, a seven-fold α-helix transmembrane domain, and an intracellular C-terminal tail. The extracellular hormone-binding region is rich in leucine repeats, responsible for binding to follicle-stimulating hormone, thereby activating signal transduction and promoting follicle development and maturation.

[0093] Isolation refers to the fact that a substance is substantially or essentially free from the components that normally accompany it in its natural state. This substance can be a cell or a macromolecule, such as a protein or nucleic acid. Isolated nucleic acids refer to polynucleotides that have been purified from sequences located laterally in their natural state, such as DNA fragments that have been removed from normally adjacent sequences.

[0094] A binding protein is a molecule or a portion thereof that binds to the target molecule FSHR. In some embodiments, the binding protein comprises an antibody. In some embodiments, the binding protein comprises an antigen-binding fragment of an antibody. In some embodiments, the binding protein may further comprise a small molecular weight component, such as a small molecule drug or a toxin. The binding protein may also be an antibody or an antigen-binding fragment thereof. In some embodiments, the binding protein comprises a ligand-binding domain of a receptor. In some embodiments, the binding protein comprises an extracellular domain of a transmembrane receptor. The binding protein may also be a ligand-binding domain of a receptor or an extracellular domain of a transmembrane receptor. In some embodiments, the binding protein comprises a single-chain antibody or a single-domain antibody. In some embodiments, the binding protein may be a single-chain antibody or a single-domain antibody.

[0095] FSHR-binding proteins may be FSHR-binding domains. In some embodiments, an FSHR-binding protein comprises an antibody that binds to FSHR or an antigen-binding fragment of an antibody. In some embodiments, an FSHR-binding protein may be an antibody or an antigen-binding fragment of an antibody. In some embodiments, an FSHR-binding protein comprises a single-chain antibody or a single-domain antibody that binds to FSHR. In some embodiments, an FSHR-binding protein may be a single-chain antibody or a single-domain antibody. In some embodiments, the single-chain antibody targeting FSHR is its ligand FSH.

[0096] An expression cassette refers to a gene sequence containing complete elements such as a promoter, coding gene, and PolyA tail, which work together to ensure effective gene expression. A linker or hinge refers to a short peptide sequence that connects different polypeptide or protein fragments, designed to maintain their respective spatial conformation and biological activity.

[0097] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An engineered immune cell, characterized in that, It expresses a chimeric antigen receptor; The chimeric antigen receptor includes an FSHR binding domain; The immune cells are selected from any one or more of the following: γδT cells, NKT cells, natural killer cells, B cells, macrophages, and DC cells.

2. The engineered immune cells according to claim 1, characterized in that, The FSHR binding domain comprises or is derived from a single-chain antibody, which comprises FSHβ of the amino acid sequence shown in SEQ ID NO:1 and FSHα of the amino acid sequence shown in SEQ ID NO:

2.

3. The engineered immune cells according to claim 1 or 2, characterized in that, The chimeric antigen receptor comprises, from its N-terminus to its C-terminus, a signal peptide, the FSHR binding domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain.

4. The engineered immune cells according to claim 3, characterized in that, The intracellular signal transduction domains originate from: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d.

5. The engineered immune cells according to claim 4, characterized in that, The intracellular signal transduction domain further includes intracellular co-stimulatory sequences.

6. The engineered immune cells according to claim 5, characterized in that, The intracellular co-stimulatory sequence is derived from any one or more of the following: CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, and CD83 ligands.

7. An engineered immune cell according to any one of claims 2-6, characterized in that, The single-chain antibody was replaced with a single-domain antibody.

8. An isolated nucleic acid molecule, characterized in that, It encodes the chimeric antigen receptor in the engineered immune cells of any one of claims 1-7.

9. An expression carrier, characterized in that, It comprises the isolated nucleic acid molecule as described in claim 8.

10. The expression vector according to claim 9, characterized in that, The expression vector is selected from any one or more of the following: lentiviral vectors, retroviral vectors, adenoviral vectors, transposons, DNA, RNA, and plasmids.

11. A method for preparing engineered immune cells, characterized in that, include: The engineered immune cells are obtained by transfecting or transducing immune cells using the expression vector described in claim 9 or 10. The immune cells are selected from any one or more of the following: γδT cells, NKT cells, natural killer cells, B cells, macrophages, and DC cells.

12. A pharmaceutical composition, characterized in that, It comprises the engineered immune cells according to any one of claims 1-7, the isolated nucleic acid molecule according to claim 8, the expression vector according to claim 9 or 10, or the engineered immune cells prepared by the method for preparing engineered immune cells according to claim 11, and a physiologically acceptable excipient.

13. The use of any one of the engineered immune cells of claims 1-7, the isolated nucleic acid molecule of claim 8, the expression vector of claim 9 or 10, or the engineered immune cells prepared by the method of preparing engineered immune cells of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating follicle-stimulating hormone-related disorders.

Images