Umbilical cord mesenchymal stem cell preparation and application thereof in ovarian disease treatment

Abstract

On one hand, the invention discloses an anti-FOLR1 single-domain antibody VHH-F01, the amino acid sequence of the VHH-F01 is as shown in SEQ ID NO: 2, and the gene sequence of the VHH-F01 is as shown in SEQ ID NO: 1. The invention also discloses an umbilical cord mesenchymal stem cell preparation, which is characterized in that the preparation introduces the single-domain antibody VHH-F01 targeting the human FOLR1 and the gene of the human Bcl-2 anti-apoptotic protein into umbilical cord mesenchymal stem cells together through a lentiviral vector to obtain a stable and high-expression cell population; vHH-F01 expressed on the membrane surface of the preparation can specifically recognize ovarian cancer cells highly expressed by FOLR1 and can be combined with the ovarian cancer cells highly expressed by FOLR1 with high affinity, and meanwhile, the survival ability of stem cells in a tumor microenvironment can be remarkably enhanced by intracellular highly expressed Bcl-2 protein. In-vitro and animal experiments show that the preparation has excellent targeting homing efficiency and remarkable tumor inhibition effect on ovarian cancer tissues, can effectively prolong the lifetime of model animals, and provides a novel efficient targeting treatment tool for cell therapy of ovarian cancer.

Description

A preparation of umbilical cord mesenchymal stem cells and its use in the treatment of ovarian diseases Technical Field

[0001] This invention belongs to the field of biomedicine and regenerative medicine technology, specifically relating to an umbilical cord mesenchymal stem cell preparation and its use in the treatment of ovarian diseases. Background Technology

[0002] Ovarian cancer is the leading cause of death among gynecological malignancies, with high-grade serous carcinoma (HGSOC) being the most common pathological type. Due to the often subtle nature of early symptoms, approximately 70% of patients are diagnosed at stage III-IV, with a 5-year survival rate of less than 30%. The standard treatment regimen is cytoreductive surgery combined with platinum-based chemotherapy; however, over 70% of patients relapse within two years and eventually develop platinum resistance, resulting in extremely poor clinical prognosis.

[0003] Current treatment for relapsed / refractory ovarian cancer faces significant bottlenecks: for platinum-sensitive relapses, platinum-based regimens can continue, but progression-free survival shortens with each additional treatment course; for platinum-resistant relapses, non-platinum monotherapy (such as poly(ADP-ribose) polymerase inhibitors and anti-angiogenic drugs) has an efficacy rate of only 10%-30%, and resistance is a common problem. Although immune checkpoint inhibitors have achieved breakthroughs in some tumors, their monotherapy response rate in ovarian cancer is low (<10%), which may be related to the immunosuppressive microenvironment of ovarian cancer.

[0004] In recent years, stem cell-based therapies have provided new ideas for the treatment of ovarian cancer. Mesenchymal stem cells (MSCs) have been explored as anticancer drug delivery carriers due to their tumor homing characteristics, secretory capacity, and potential for engineerability. Preclinical studies have shown that infused MSCs can migrate to ovarian cancer lesions and inhibit tumor growth by delivering apoptosis-inducing factors (such as TRAIL) or expressing anticancer genes. However, existing stem cell therapies face key challenges in the treatment of ovarian cancer: (1) insufficient targeting efficiency, with only a very small percentage of cells (<1%) reaching the tumor site after intravenous infusion, limiting efficacy and potentially increasing the risk of off-target effects; (2) constraints from the tumor microenvironment (TME), with factors such as hypoxia, acidosis, and immune cell infiltration in the ovarian cancer TME leading to short survival time and difficulty in maintaining function of transplanted stem cells.

[0005] To overcome these obstacles, existing research has attempted to engineer MSCs. For example, some patents report using gene editing technology to overexpress MSCs homing-related receptors such as CXCR4, thereby enhancing their migration ability to lesions; other studies have combined MSCs with targeting antibodies or exosomes to improve the precision of treatment. However, these strategies still have problems such as insufficient targeting specificity and therapeutic efficacy, complex preparation processes, or potential safety risks.

[0006] Therefore, developing a novel stem cell preparation that can efficiently and specifically target ovarian cancer tissue, maintain functional activity in the complex tumor microenvironment, and possess a clear mechanism of action and good formulation stability is of great clinical significance and application value for overcoming the current treatment bottleneck of ovarian cancer. Summary of the Invention

[0007] To address the above technical problems, this invention discloses an umbilical cord mesenchymal stem cell preparation and its use in the treatment of ovarian diseases.

[0008] Therefore, this invention discloses an anti-FOLR1 single-domain antibody VHH-F01, the amino acid sequence of which is shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding VHH-F01 is shown in SEQ ID NO:1; the recombinant prokaryotic expression vector containing the gene described in SEQ ID NO:1 uses pET-28a+ as a backbone, inserts the VHH-F01 gene, and the C-terminus of VHH-F01 in the vector has a His tag.

[0009] In one aspect, the present invention also discloses a genetically engineered human umbilical cord-derived mesenchymal stem cell, FOLR1-VHH-Bcl-2-UC-MSCs, wherein the FOLR1-VHH-Bcl-2-UC-MSCs stably express tandem elements, wherein the tandem elements are, in sequence, an EcoRI site, a CMV promoter, a CD8α signal peptide, the VHH-F01 of claim 1, a PDGFR transmembrane domain, a P2A peptide, human Bcl-2 protein, a T2A peptide, GFP, and an XhoI site. The nucleotide sequence of the CMV promoter is shown in SEQ ID NO:3. The amino acid sequence of the CD8α signal peptide is MALPVTALLLPLALLLHAARP; the amino acid sequence of the PDGFR transmembrane domain is VAAAVLVLLVIVIISLIVLVVIW. The amino acid sequence of the P2A peptide is GSGATNFSLLKQAGDVEENPGP; the amino acid sequence of the T2A peptide is EGRGSLLTCGDVEENPG. The nucleotide sequence of the human Bcl-2 protein is shown in SEQ ID NO:4. The nucleotide sequence of the GFP is shown in SEQ ID NO:5.

[0010] In one aspect, the present invention also discloses the application of the VHH-F01 in the preparation of FOLR1 positive tumor diagnostic reagents or therapeutic drugs.

[0011] In one aspect, the present invention also discloses the application of the FOLR1-VHH-Bcl-2-UC-MSCs in the preparation of FOLR1-positive tumor therapeutic drugs.

[0012] The beneficial effects of this invention are summarized as follows:

[0013] (1) VHH-F01 has significant advantages: First, it has high specificity, binding only to FOLR1 and not cross-reacting with irrelevant antigens such as EGFR and BSA, which can effectively avoid off-target effects and ensure the accuracy of targeting; Second, it has strong affinity, with an equilibrium dissociation constant of 8.2×10^-11 M, which is 70 times that of the existing commercial antibody M9346A, and the dissociation rate is slow, which can maintain the binding state with tumor cells for a long time and improve the duration of local drug action in tumors; Third, it has a small molecular weight, with a molecular weight of about 15kDa, which significantly improves tissue penetration and makes it easier to penetrate the tumor stroma and enter the tumor, solving the problem of poor tissue penetration of traditional monoclonal antibodies; Fourth, it is easy to produce and purify, and can achieve high-efficiency expression by relying on the prokaryotic expression system. The product with a purity of not less than 95% can be obtained by Ni-NTA affinity chromatography. The production cost is low and the yield is stable, which is conducive to large-scale production and clinical translation.

[0014] (2) FOLR1-VHH-Bcl-2-UC-MSCs have multiple advantages in terms of function and therapeutic effect: On the one hand, through the dual functional design of VHH-F01-mediated targeted binding and Bcl-2-inhibited apoptosis, the core problems of low targeted homing efficiency and easy apoptosis in wild-type mesenchymal stem cells in the tumor microenvironment are solved simultaneously, achieving synergistic enhancement of function; on the other hand, the targeted enrichment efficiency is high. In in vitro experiments, the binding rate of FOLR1-overexpressing ovarian cancer cells OVCAR-3 reached 68.3%, which is 7.4-7.9 times that of wild-type mesenchymal stem cells. In in vivo experiments, the tumor site was enriched within 72 hours. The concentration of the collected cells was 15.3 times that of the control group, which significantly increased the local concentration of cells for tumor treatment. Simultaneously, the treatment effect was excellent, with a tumor inhibition rate of 62.0% at 21 days, 3.1 times that of wild-type mesenchymal stem cells. The median survival time in nude mice was extended to 56.7 days, 74.5% longer than the PBS group, and the 180-day survival rate reached 25%, with some individuals achieving long-term survival, significantly superior to existing cell therapy protocols. Furthermore, the safety profile was good; in vivo distribution experiments showed no abnormal accumulation in normal organs such as the liver, spleen, lungs, and kidneys. The weight loss rate of animals during treatment was only -2.0%, and there were no cases of termination of the experiment due to toxic side effects, ensuring the safety of clinical application.

[0015] (3) From the perspective of application prospects, VHH-F01 can be used alone as a diagnostic reagent for targeted imaging or ELISA detection of FOLR1 positive tumors, and can also be used as a target element of antibody drug conjugate and targeted drug delivery system to expand the application scenarios of treatment; FOLR1-VHH-Bcl-2-UC-MSCs can not only be used directly for the treatment of FOLR1 positive ovarian cancer, but can also be further expanded to the treatment of other FOLR1 positive tumors by loading chemotherapy drugs, siRNA, oncolytic viruses, etc., providing diversified strategies for precision treatment of tumors, and has broad clinical application value and industrialization potential. Attached Figure Description

[0016] Figure 1 shows the SDS-PAGE detection results of VHH-F01, where 1 represents VHH-F01.

[0017] Figure 2. Results of double enzyme digestion identification of plasmid, where 1 is the plasmid.

[0018] Figure 3. Observation results under fluorescence microscopy.

[0019] Figure 4. Survival curve. Detailed Implementation

[0020] Unless otherwise defined, 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0021] Unless otherwise specified, the reagents, methods, and equipment used in this 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.

[0022] Example 1: Preparation and testing of anti-FOLR1 single-domain antibody (VHH-F01)

[0023] 1. Experimental Materials

[0024] 1.1 Biological materials: 6-month-old healthy alpacas; human FOLR1 recombinant antigen (MCE, HY-P70296); FOLR1-overexpressing ovarian cancer cell line OVCAR-3 and FOLR1-low-expressing ovarian cancer cell line SKOV-3; commercially available anti-FOLR1 monoclonal antibody M9346A (MCE, HY-P99225).

[0025] 1.2 Vectors and strains: prokaryotic expression vector pET-28a (+); phage display vector pComb3X; Escherichia coli TG1 (for phage library construction) and BL21 (DE3) (for protein expression).

[0026] 1.3 Reagents: Freund's complete / incomplete adjuvant (Sigma); RNA extraction kit (TAKARA); reverse transcription kit (Thermo); PCR high-fidelity enzyme (NEB); Ni-NTA affinity chromatography column (GE Healthcare); ELISA kit; SPR chip (CM5, Biacore); MTT cell proliferation assay kit (Solarbio).

[0027] 2. Experimental Procedures and Results

[0028] 2.1 Immunology and VHH Gene Bank Construction

[0029] 2.1.1 Immunization Protocol: Human FOLR1 recombinant antigen (50 μg / animal) was emulsified with an equal volume of Freund's complete adjuvant and administered via subcutaneous injection at multiple sites in llamas (day 0). Booster immunizations were performed on days 14 and 28 using the same dose of antigen emulsified with Freund's incomplete adjuvant. On day 42, a booster immunization was performed via tail vein injection of unadjuvanted antigen (50 μg / animal), and 50 mL of peripheral blood was collected 72 hours later. Results showed that on day 42, the serum anti-FOLR1 antibody titer of the llamas reached 1:10^5 (ELISA detection), indicating successful immunization.

[0030] 2.1.2 Lymphocyte isolation and RNA extraction: Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium, and total RNA was extracted according to the RNA extraction kit instructions. The RNA purity was detected by Nanodrop (A260 / A280 = 1.8~2.0).

[0031] 2.1.3 cDNA synthesis and VHH gene amplification: cDNA was synthesized by reverse transcription using total RNA as a template and Oligo (dT) primers; PCR amplification was performed using alpaca VHH-specific primers (98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles, 72℃ final extension for 5 min); VHH fragments were recovered by agarose gel electrophoresis.

[0032] 2.2 Construction and Screening of Phage Display Libraries

[0033] 2.2.1 Library Construction: The VHH gene was ligated into the pComb3X vector, which had been double-digested with EcoRI / XhoI, and transformed into *E. coli* TG1 competent cells. The cells were plated on LB-Amp plates and incubated at 37°C for 16 h. All colonies were collected, and LB medium containing helper phage M13KO7 was added. The cells were incubated at 37°C with shaking for 12 h. The supernatant was collected by centrifugation, which was the VHH phage display library. The library size (≥10^8 CFU) and recombination rate (≥90%, verified by colony PCR) were determined. The results showed that the VHH phage display library had a size of 2.3 × 10^8 CFU and a recombination rate of 92%, meeting the screening requirements.

[0034] 2.2.2 Affinity Screening: Dilute human FOLR1 antigen to 10 μg / mL with 0.05 M carbonate buffer (pH 9.6), coat ELISA plates (100 μL / well) at 4°C overnight; discard the coating solution, block with PBST (0.05% Tween-20) containing 5% skim milk for 2 h (37°C); add phage library (100 μL / well), incubate at 37°C for 1 h, wash with PBST 10 times (round 1), 15 times (round 2), and 20 times (round 3) to gradually increase the screening stringency; add 0.2 M Glycine-HCl (pH 2.2) to elute the bound phage, and neutralize with 1 M Tris-HCl (pH 9.1) after 10 min; infect TG1 with the eluted phage, amplify and use for the next round of screening, for a total of 3 rounds of screening.

[0035] 2.3 Expression and purification of VHH-F01

[0036] 2.3.1 Identification of positive clones: Single colonies after the third round of screening were picked and subjected to phage ELISA detection (coated with FOLR1 antigen, HRP-labeled anti-M13 antibody was used to detect binding activity). Positive clones with OD450nm≥2.0 were selected and sequenced to obtain the VHH-F01 gene sequence (as shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2).

[0037] 2.3.2 Prokaryotic expression: The VHH-F01 gene was cloned into the pET-28a (+) vector (with a His tag at the C-terminus) and transformed into BL21 (DE3). Single colonies were picked and inoculated into LB-Kana medium and cultured at 37°C until OD600nm=0.6. IPTG (final concentration 0.5mM) was added and expression was induced at 25°C for 16h. The cells were collected by centrifugation, resuspended in lysis buffer (50mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0), and sonicated (300W power, 3s operation, 5s interval, 30min total). The supernatant was collected by centrifugation.

[0038] 2.3.3 Affinity purification: The supernatant was loaded onto a Ni-NTA chromatography column, and impurities were eluted with washing buffer containing 20 mM imidazole, and the target protein was eluted with elution buffer containing 250 mM imidazole. The protein was dialyzed with PBS (pH 7.4) for 24 h (with 3 buffer changes), and the purity was determined by SDS-PAGE (as shown in Figure 1, the molecular weight is approximately 15 kDa, ≥95%). The protein concentration (5.2 mg / mL) was determined by the BCA method.

[0039] 2.4 Verification of VHH-F01

[0040] 2.4.1 Specificity test (indirect ELISA)

[0041] (1) Experimental procedure: The ELISA plates were coated with 10 μg / mL human FOLR1, human EGFR (irrelevant antigen), and bovine serum albumin (BSA) and incubated overnight at 4°C; blocked with 5% skim milk-PBST for 2 h (37°C); VHH-F01 or M9346A at gradient concentrations (0.1, 0.5, 1, 5, 10 μg / mL) were added and incubated at 37°C for 1 h; HRP-labeled anti-His antibody (for VHH-F01) or anti-human IgG antibody (for M9346A) were added and incubated at 37°C for 1 h; TMB was used for color development for 15 min, the reaction was terminated with 2M H2SO4, and the OD450nm value was measured.

[0042] (2) The results showed (Table 1): VHH-F01 and M9346A both specifically bind to human FOLR1 and do not bind to irrelevant antigens, and their specificity is consistent.

[0043] Table 1. Specificity test results (OD450nm value)

[0044]

[0045] 2.4.2 Affinity Test (SPR)

[0046] (1) Experimental procedure: Human FOLR1 antigen was immobilized on the surface of CM5 chip by amine coupling method, with an immobilization density of about 1000RU; VHH-F01 or M9346A was diluted with running buffer (PBST) to 0.1, 0.3, 1, 3, 10 nM, and flowed through the chip at a flow rate of 30 μL / min, and the binding phase (120s) and dissociation phase (300s) were recorded; a 1:1 binding model was fitted using Biacore T200 software and the equilibrium dissociation constant (KD) was calculated.

[0047] (2) The results show (Table 2): the KD value of VHH-F01 is significantly lower than that of M9346A (P<0.01), and the affinity is 70 times that of M9346A, mainly due to the slower dissociation rate.

[0048] Table 2 Affinity Test Results

[0049]

[0050] 2.4.3 Cell binding activity assay (FACS)

[0051] (1) Experimental procedure: Collect OVCAR-3 (FOLR1 high expression) and SKOV-3 (FOLR1 low expression) cells in the log phase and adjust the concentration to 1×10^6 cells / mL; add VHH-F01 or M9346A (final concentration 10μg / mL) and incubate at 4℃ for 30min; wash twice with PBS, add FITC-labeled anti-His antibody or anti-human IgG antibody and incubate at 4℃ for 20min; detect fluorescence signal by flow cytometry and calculate positive cell rate and mean fluorescence intensity (MFI).

[0052] (2) The results showed (Table 3): In FOLR1-overexpressing OVCAR-3 cells, the positive cell rate of VHH-F01 was not significantly different from that of M9346A, but MFI was higher; there was no obvious binding in SKOV-3 cells, and the cell-level specificity was consistent.

[0053] Table 3 Results of cell binding activity test

[0054]

[0055] 2.4.4 MTT assay for cell proliferation inhibition

[0056] (1) Experimental procedure: OVCAR-3 cells were seeded at 5×10^3 cells / well into 96-well plates and cultured at 37℃ and 5% CO2 for 24h; VHH-F01 or M9346A at gradient concentrations (0.01, 0.1, 1, 10, 100 μg / mL) or M9346A were added, with 3 replicates per group, and cultured for another 48h; 20 μL of MTT solution (5 mg / mL) was added to each well and cultured for 4h; the supernatant was discarded, 150 μL of DMSO was added, the mixture was shaken for 10 min, the OD570nm value was measured, and the cell proliferation inhibition rate and half-maximal inhibitory concentration (IC50) were calculated; where the cell proliferation inhibition rate = (1 - mean OD570nm value of experimental group / mean OD570nm value of blank control) × 100%.

[0057] (2) The results showed that the IC50 of VHH-F01 was significantly lower than that of M9346A (P<0.01), and it had a stronger inhibitory activity on the proliferation of OVCAR-3 cells; there was no significant difference in the inhibition rate between the two at high concentrations (P>0.05).

[0058] Table 4 Results of cell proliferation inhibition activity test

[0059]

[0060] Example 2: Preparation and testing of FOLR1-VHH-Bcl-2-UC-MSCs

[0061] 1. Construction of gene expression vector

[0062] 1.1 Element tandem and whole gene synthesis: Tandem expression cassettes were designed based on the sequences of each element (EcoRI and XhoRI were introduced as restriction sites, as shown in Table 5); the synthesis was completed by Genscript Biotech according to the design requirements.

[0063] Table 5. Composition of each component in the cascaded expression frame.

[0064]

[0065] 1.2 Connection between the expression frame and the carrier skeleton

[0066] 1.2.1 Double digestion of vector and expression cassette

[0067] (1) Reaction system (50 μL): pLVX-PGK-Puro vector (1 μg) or synthetic expression cassette (1 μg), EcoRI (1 μL), XhoRI (1 μL), 10×CutSmart Buffer (5 μL), ddH2O to 50 μL;

[0068] (2) Enzyme digestion conditions: incubation at 37℃ for 3 hours, followed by heat inactivation at 65℃ for 20 minutes;

[0069] (3) Recovery of enzyme digestion products: 1% agarose gel electrophoresis was used to separate the products, and the products were purified using a recovery kit. The concentration of the vector backbone was measured to be 200 ng / μL and the concentration of the expression cassette was 300 ng / μL.

[0070] 1.2.2 Connection Reaction

[0071] (1) Ligation system (20 μL): linearized vector backbone (50 ng), synthesized expression cassette (150 ng, vector: insert molar ratio = 1:3), 10×T4 ligation buffer (2 μL), T4 DNA ligase (1 μL), ddH2O added to 20 μL;

[0072] (2) Connection conditions: incubate at 16℃ for 16h, then inactivate at 65℃ for 10min.

[0073] 1.2.3 Transformation and positive clone screening: 10 μL of the ligation product was transformed into 50 μL of Escherichia coli DH5α competent cells, incubated on ice for 30 min, then heat-shocked at 42℃ for 45 s, then incubated on ice for 2 min. 500 μL of LB medium was added, and the cells were incubated at 37℃ for 1 h. 200 μL of the bacterial culture was plated on LB-ampicillin plates (100 μg / mL) and incubated at 37℃ for 16 h. A total of 28 single colonies were obtained (ligation efficiency: 28 colonies / μg vector).

[0074] 1.3 Identification of positive clones and vector quality testing

[0075] 1.3.1 Enzyme digestion verification: Plasmids from the above 10 positive clones were extracted and digested with EcoRI / XhoI (37℃, 2h). 1% agarose gel electrophoresis showed two bands (Figure 2): approximately 8.1kb (vector backbone) and approximately 2.6kb (expression cassette). The enzyme digestion accuracy was 100% (10 / 10).

[0076] 1.3.2 Full Sequence Validation: Three clones with correct restriction enzyme digestion were selected for full sequence determination (BGI Genomics). The results showed that the sequences of each element were consistent with the design, with no base mutations; the restriction enzyme sites were correctly aligned; and the reading frames were continuous.

[0077] 1.3.3 Vector quality testing: Clone No. 1 was selected for amplification culture, plasmid was extracted, concentration was 1.5 μg / μL (Nanodrop), A260 / A280=1.89; the endotoxin content was determined by the Limulus Amebocyte Lysate (LAL) reagent method to be 0.015 EU / μg, which meets the requirements for lentivirus packaging (<0.1 EU / μg).

[0078] 2. Isolation and culture of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)

[0079] 2.1 Isolation of umbilical cord mesenchymal stem cells (refer to the method of extraction and isolation in CN201910096096.9): Wharton's jelly tissue was isolated from the umbilical cord of a healthy full-term pregnancy. Primary UC-MSCs were cultured using the tissue block adherence method. Serum-free culture medium with serum substitute was used to avoid contamination by animal-derived components.

[0080] 2.2 Take third-generation hUC-MSCs, digest them with 0.25% trypsin (containing EDTA) (37℃, 3 min), count them with a counting chamber, and the cell viability (trypan blue staining) is 96.5%; adjust the cell concentration to 5×10^4 cells / mL, seed them in T75 culture flasks, change the medium after 24 h to remove non-adherent cells, and after 48 h the cell confluence reaches 70%-80% for transduction.

[0081] 3 Lentiviral Packaging and Transduction

[0082] 3.1 Materials: Packaging cells: 293T cells; Packaging plasmids: pSPAX2 (gag / pol) and pMD2.G (VSV-G);

[0083] Polybrene (final concentration 8 μg / mL) was used as a transduction reagent.

[0084] 3.2 Steps

[0085] 3.2.1 Lentiviral Packaging: 293T cells were seeded in 10cm culture dishes (2×10^6 cells / dish) and cultured for 24h until confluence reached 70%; the full-length vector plasmid constructed above was co-transfected with Lipofectamine 3000 (10μg) + pSPAX2 (7.5μg) + pMD2.G (5μg), and the medium was changed after 6h; the supernatant was collected at 48h and 72h after transfection, filtered through a 0.45μm filter membrane, and concentrated by ultracentrifugation (25,000rpm, 4℃, 2h), and the titer was determined to be 1.2×10^8 TU / mL (GFP fluorescence method).

[0086] 3.2.2 hUC-MSC transduction: hUC-MSCs were seeded at 2×10^5 cells / well in 6-well plates and cultured for 24 h; lentivirus (MOI=10) + Polybrene (8 μg / mL) were added and incubated at 37℃ for 12 h, then the medium was changed; after 72 h of transduction, GFP expression was observed under a fluorescence microscope, and the initial transduction efficiency was 42.3% (ImageJ count).

[0087] 4. Puromycin screening and flow cytometry

[0088] 4.1 Puromycin screening: 72 h after transduction, the medium was replaced with one containing 2 μg / mL puromycin; the medium was changed every 3 days and the screening was continued for 2 weeks. The proportion of surviving cells decreased from the initial 42.3% to 18.5% (day 7) and finally stabilized at 15.2% (day 14). All of them were GFP positive (observed under a fluorescence microscope, Figure 3).

[0089] 4.2 Flow cytometry cell sorting: After screening, cells were digested with trypsin, washed twice with PBS, and the concentration was adjusted to 1×10^6 cells / mL; GFP-positive cells were sorted by flow cytometer (excitation wavelength 488nm, emission wavelength 530nm), and the sorting purity was set to "high purity mode"; the GFP positivity rate after sorting was 98.7%, and the cell viability was 92.3%.

[0090] 5. Expanded culture and cryopreservation

[0091] 5.1 The sorted GFP-positive cells were seeded into T75 culture flasks and cultured in α-MEM containing 10% FBS. The cells were passaged once every 3-4 days. The cell number reached 5×10^7 by the 3rd passage.

[0092] 5.2 Cryopreservation solution formulation: α-MEM + 20% FBS + 10% DMSO, dispensed into cryovials at a rate of 1×10^6 cells / vial;

[0093] 5.3 Programmed cooling: 4℃ for 30 min → -20℃ for 2 h → -80℃ overnight → transfer to liquid nitrogen (-196℃) for storage;

[0094] 5.4 Validation of cryopreservation and thawing: Three cryopreserved tubes were randomly selected for thawing. After 24 hours, the cell adhesion rate was 90.5%, the GFP positivity rate remained at 98.1%, and the cell viability was 95.2%. The cells were labeled as FOLR1-VHH-Bcl-2-UC-MSCs and stored in liquid nitrogen for later use.

[0095] Example 3: In vitro targeting experiment

[0096] 1 Experimental Methods

[0097] 1.1 Materials: OVCAR-3 (FOLR1 high expression), SKOV-3 (FOLR1 low expression), FOLR1-VHH-Bcl-2-UC-MSCs (experimental group, GFP labeled), wild-type UC-MSCs (control group 1, unlabeled), empty vector transduced UC-MSCs (control group 2, GFP labeled, transduced empty pLVX-PGK-Puro); RPMI-1640 medium (Gibco) + 10% fetal bovine serum (FBS), 4% paraformaldehyde, DAPI staining solution (1 μg / mL), PBS (pH 7.4); 24-well cell culture plates (Corning), fluorescence microscope, flow cytometer, cell counting chamber, etc.

[0098] 1.2 Experimental Procedure

[0099] 1.2.1 Cell pretreatment and seeding: OVCAR-3 / SKOV-3 cells were digested with 0.25% trypsin (37℃, 3 min), and the concentration was adjusted to 2×10⁻⁶ cells after counting. 4 Cells / well were seeded into 24-well plates (500 μL of complete culture medium per well) and incubated at 37°C and 5% CO2 for 24 h, ensuring cell adhesion >90% (microscopic observation). UC-MSCs from each group were digested, counted, and their concentration adjusted to 1 × 10⁻⁶. 5 cells / well (to tumor cell ratio 1:5), for later use.

[0100] 1.2.2 Co-culture and washing: Discard the old culture medium for tumor cells in the 24-well plate, add 500 μL of complete culture medium containing UC-MSCs to each well, and co-culture at 37℃ and 5% CO2 for 2 h; after co-culture, add 5 mL of PBS (room temperature) to each well, gently shake for 1 min and then discard, repeat 3 times to ensure the removal of unbound free UC-MSCs (take the supernatant after the third wash, and flow cytometry to confirm that free cells < 0.5%).

[0101] 1.2.3 Fixation and staining: Add 400 μL of 4% paraformaldehyde to each well, fix at room temperature for 15 min, aspirate and wash twice with PBS (3 mL each time); add 200 μL of DAPI staining solution to each well, stain at room temperature in the dark for 10 min, wash twice with PBS, and observe under an inverted fluorescence microscope.

[0102] 1.2.4 Imaging and Quantitative Analysis

[0103] (1) Fluorescence microscopy observation: excitation wavelengths of 488nm (GFP) and 350nm (DAPI) were used. Five fields of view were randomly selected from each well under a 40× objective lens and photographed. The "GFP" count was performed using ImageJ software. + UC-MSCs and DAPI + Tumor cell overlap / total DAPI + "Tumor cell count" is used to calculate the binding rate;

[0104] (2) Flow cytometry quantification: Cells in 24-well plates were digested with trypsin (to avoid damaging bound UC-MSCs), collected into flow cytometry tubes, washed twice with PBS, and the concentration was adjusted to 1×10⁻⁶. 6 GFP was detected by flow cytometry in cells / mL. + Cell ratio (excitation 488nm, emission 530nm), each group has 3 replicates, and each experiment is repeated 3 times independently.

[0105] 2. Experimental Results

[0106] 2.1 The binding rate of OVCAR-3 in the experimental group (68.3%) was significantly higher than that in control group 1 (8.7%) and control group 2 (9.2%), with a highly statistically significant difference (t=28.6, P<0.001), and the binding efficiency was 7.4-7.9 times that of the control group; the binding rate of SKOV-3 in each group was <5%, and there was no statistically significant difference between groups (F=0.32, P>0.05), excluding non-specific binding; the GFP of OVCAR-3 in the experimental group... + The cell MFI (2586) was 7.3–7.9 times that of the control group, indicating that a greater number of UC-MSCs were bound to a single tumor cell, further validating the targeted binding ability. See Table 6 for details.

[0107] Table 6. In vitro binding rate and mean fluorescence intensity (MFI) analysis (n=3, mean ± standard deviation)

[0108]

[0109] Example 4: In vivo targeting experiment

[0110] 1 Experimental Methods

[0111] 1.1 Materials: 6-8 week old female BALB / c nude mice (weight 18-22g); OVCAR-3-Luc cells (stable expression of luciferase, constructed in our laboratory), DIR fluorescent dye (1mg / mL), sterile PBS, isoflurane (anesthetic); IVIS LuminaXR in vivo imaging system, vernier calipers, electronic balance, sterile surgical instruments (ophthalmic scissors / forceps), etc.

[0112] 1.2 Experimental Procedure

[0113] 1.2.1 Establishment of an orthotopic ovarian cancer model: Nude mice were fasted for 6 hours before surgery and anesthetized with isoflurane inhalation (1.5%-2% concentration, oxygen flow rate 1L / min); a midline abdominal incision (approximately 0.5cm) was made to expose the left ovary, and 2×10⁻⁶ ovaries were inserted using a 27G needle. 6 OVCAR-3-Luc cells (resuspended in 100 μL sterile PBS) were slowly injected under the ovarian capsule, and the incision was sutured (5-0 absorbable sutures). Postoperatively, the patient was housed individually with free access to food and water. Starting on day 7 after modeling, tumor luciferase activity was detected weekly using an in vivo imaging system (D-luciferin potassium salt 150 mg / kg injected intraperitoneally, imaging 10 min later). Simultaneously, the long diameter (L) and short diameter (W) of the tumor were measured using the formula V = 0.5 × L × W. 2 Calculate the volume when the tumor volume reaches 150 mm. 3 The experiment began approximately 14 days after modeling.

[0114] 1.2.2 Labeling and grouping of UC-MSCs: Each group of UC-MSCs (1×10⁻⁶) 7 DIR dye (final concentration 5 μM) was added to cells / mL and incubated at 37 °C for 30 min. The cells / mL were washed three times with PBS (centrifuged at 800 × g for 5 min each time) to remove free dye. The labeling efficiency was > 95% by flow cytometry. Nude mice were randomly divided into two groups (n=6 / group). The experimental group was injected with DIR-labeled FOLR1-VHH-Bcl-2-UC-MSCs, and the control group was injected with DIR-labeled wild-type UC-MSCs.

[0115] 1.2.3 Injection and Imaging: Each nude mouse was slowly injected via the tail vein with 100 μL of a solution containing 1×10⁻⁶ ppm. 6UC-MSCs were infused with PBS to ensure no leakage. In vivo imaging was performed on anesthetized nude mice at 24h, 48h, and 72h post-injection (DIR excitation wavelength 748nm, emission wavelength 780nm, exposure time 10s, field of view 12.5cm, f / stop 2). Fluorescence intensity at tumor sites was quantified using IVIS software (unit: ×10). 8 photons / s / cm 2 / sr); after 72 hours of in vivo imaging, nude mice were euthanized (with an overdose of isoflurane), and ovarian tumors, liver, spleen, lungs, and kidneys were aseptically dissected and removed. The organs were rinsed three times with PBS (to remove residual blood), and the moisture was absorbed with filter paper. The organs were then imaged under the same conditions, and the fluorescence intensity of each organ was quantified.

[0116] 2. Experimental Results

[0117] The DIR labeling rate of UC-MSCs in all groups was 96.2% ± 1.5% (flow cytometry detection), with no difference between groups; 14 days after modeling, the tumor volume of all nude mice was 150.3 ± 12.5 mm. 3 The luciferase activity was 2.8 × 10⁻⁶. 6 ±0.5×10 6 The fluorescence intensity at the tumor site was measured in photons / s to ensure model uniformity. In the experimental group, the fluorescence intensity at the tumor site increased over time (2.9-fold increase from 24h to 72h), while the control group showed no significant change, indicating that FOLR1-VHH-mediated UC-MSCs can continuously accumulate at the tumor site. At 72h, the fluorescence intensity of the tumor in the experimental group was 15.8 times that of the control group (P<0.001), while there was no difference in fluorescence intensity among the groups for organs such as the liver, spleen, lung, and kidney (P>0.05), excluding non-specific organ distribution. The liver fluorescence intensity was slightly higher (8.2 in the experimental group and 9.5 in the control group), consistent with the normal liver clearance pathway of MSCs in vivo, with no abnormal accumulation, demonstrating good safety of the targeting process. Details are shown in Tables 7 and 8.

[0118] Table 7 Dynamic changes in fluorescence intensity at tumor sites in vivo (×10) 8 photons / s / cm 2 / sr, n=6, mean ± standard deviation)

[0119]

[0120] Table 8. Fluorescence intensity of isolated organs after 72 hours (×10) 8 photons / s / cm 2 (n=6, mean ± standard deviation)

[0121]

[0122] Example 5: Evaluation of Treatment Efficacy

[0123] 1 Experimental Methods

[0124] 1.1 Materials and Grouping

[0125] 1.1.1 Animal model: Same as Example 4 (OVCAR-3-Luc orthotopic model, tumor volume 150 mmHg) 3 );

[0126] 1.1.2 Grouping: Nude mice were randomly divided into 3 groups (n=8 / group): PBS group (injected with 100μL sterile PBS), wild-type UC-MSCs group (injected with 1×10⁻⁶ ppm of sterile PBS), and wild-type UC-MSCs group (injected with 1×10⁻⁶ ppm of sterile PBS). 6 Wild-type UC-MSCs), experimental group (injected with 1×10 6 FOLR1-VHH-Bcl-2-UC-MSCs);

[0127] 1.2 Experimental Procedure

[0128] 1.2.1 Treatment regimen: Once a week for 3 consecutive weeks (days 0, 7, and 14), with an injection volume of 100 μL per mouse. Observe for 5 minutes after injection to ensure no allergic reaction (such as rapid breathing or convulsions). All nude mice were housed in an SPF-grade animal room (temperature 22±2℃, humidity 50%±5%, 12-hour light-dark cycle) and had free access to sterilized feed and water.

[0129] 1.2.2 Monitoring Indicators and Standards: Measure the volume (using calipers) at the same time every 3 days and calculate the volume. When the tumor volume is ≥2000 mm², the tumor is considered to be at risk. 3 When ulcers appear, the mouse is considered to be in a near-death state. It is weighed at the same time every 3 days (using an electronic balance). If the weight decreases by more than 20% from the initial value, it is considered abnormal (the experiment is terminated). Starting from the first treatment (day 0), the survival time of each nude mouse is recorded until the near-death state (humane endpoint), and a Kaplan-Meier survival curve is plotted. On day 21 of treatment, D-fluorescein potassium salt is injected intraperitoneally, and in vivo imaging is used to detect tumor luciferase activity and quantify tumor burden.

[0130] 2. Experimental Results

[0131] The tumor inhibition rate in the experimental group after 21 days of treatment (62.0%) was 3.1 times that of the wild-type group (20.2%), and the tumor fluorescence activity was only 22.7% of that in the PBS group, demonstrating that FOLR1 targeting significantly enhanced the tumor-inhibiting ability of UC-MSCs. The body weight change rate in the experimental group (-2.0%) was significantly lower than that in the PBS group (-8.5%), and no nude mice discontinued the experiment due to weight loss, indicating that the treatment had no obvious toxic side effects. The median survival time in the experimental group was prolonged by 74.5%, and long-term survival individuals were observed, suggesting that targeting UC-MSCs not only inhibits tumor growth but also delays disease progression and improves prognosis. Details are shown in Tables 9 and 10.

[0132] Table 9. Changes in tumor volume and body weight during treatment (n=8, mean ± standard deviation)

[0133]

[0134] Table 10 Tumor burden and survival data at 21 days after treatment

[0135]

[0136] Survival curves (Figure 4) showed that the median survival of nude mice in the experimental group (56.7 days) was significantly longer than that in the wild-type UC-MSCs group (38.2 days, P<0.01) and the PBS group (32.5 days, P<0.001), with a 180-day survival rate of 25% (2 / 8 of the experimental group survived), while all mice in the PBS group and the wild-type group died within 60 days.

[0137] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-domain antibody against FOLR1, VHH-F01, characterized in that, The amino acid sequence of VHH-F01 is shown in SEQ ID NO:

2.

2. A gene encoding VHH-F01 as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. A recombinant prokaryotic expression vector containing the gene of claim 2, characterized in that, The vector uses pET-28a+ as a backbone and inserts the VHH-F01 gene, and the C-terminus of VHH-F01 in the vector has a His tag.

4. A genetically engineered human umbilical cord-derived mesenchymal stem cell (FORLR1-VHH-Bcl-2-UC-MSCs), characterized in that, The FOLR1-VHH-Bcl-2-UC-MSCs stably express tandem elements, wherein the tandem elements are, in sequence, EcoRI site, CMV promoter, CD8α signal peptide, VHH-F01 as described in claim 1, PDGFR transmembrane domain, P2A peptide, human Bcl-2 protein, T2A peptide, GFP, and XhoRI site.

5. The FORLR1-VHH-Bcl-2-UC-MSCs according to claim 4, characterized in that, The nucleotide sequence of the CMV promoter is shown in SEQ ID NO:

3.

6. The FORLR1-VHH-Bcl-2-UC-MSCs according to claim 4, characterized in that, The amino acid sequence of the CD8α signal peptide is MALPT VALLLPLALLLHAARP; the amino acid sequence of the PDGFR transmembrane domain is VAAAVLVLLVIVIISLIVLVVIW; the amino acid sequence of the P2A peptide is GSGATNFSLLKQAGDVEENPGP; and the amino acid sequence of the T2A peptide is EGRGSLLTCGDVEENPG.

7. The FORLR1-VHH-Bcl-2-UC-MSCs according to claim 4, characterized in that, The nucleotide sequence of the human Bcl-2 protein is shown in SEQ ID NO:

4.

8. The FORL1-VHH-Bcl-2-UC-MSCs according to claim 4, characterized in that, The nucleotide sequence of the GFP is shown in SEQ ID NO:

5.

9. The use of VHH-F01 as described in claim 1 in the preparation of FOLR1 positive tumor diagnostic reagents or therapeutic drugs.

10. The use of the FORL1-VHH-Bcl-2-UC-MSCs of claim 4 in the preparation of a FORL1-positive tumor therapeutic drug.

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