CN114788877B - Genetically engineered hematopoietic stem cell drug delivery system and preparation method and application thereof - Google Patents
Genetically engineered hematopoietic stem cell drug delivery system and preparation method and application thereof Download PDFInfo
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- CN114788877B CN114788877B CN202210312766.8A CN202210312766A CN114788877B CN 114788877 B CN114788877 B CN 114788877B CN 202210312766 A CN202210312766 A CN 202210312766A CN 114788877 B CN114788877 B CN 114788877B
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- A61P35/04—Antineoplastic agents specific for metastasis
Abstract
The invention discloses a genetically engineered hematopoietic stem cell drug delivery system for treating bone metastasis. The invention targets bone marrow sites by mediating hematopoietic stem cells to overexpress immunotherapeutic substances and loading a drug TGF-beta inhibitor, and is used for treating bone marrow metastasis. The genetically engineered hematopoietic stem cell drug delivery system has the natural property of targeting bone marrow, low immunogenicity and good biological safety, can be produced by cell culture expansion after genetic modification, is convenient to prepare and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a genetically engineered hematopoietic stem cell drug delivery system, a preparation method and application thereof.
Background
Bone is one of the most common metastasis destinations in breast, prostate and lung cancers. More than two-thirds of bone metastases are not limited to bone, but rather are further transferred to systemic metastases, ultimately leading to patient death. In addition, bone metastases are often accompanied by bone destruction, which can lead to severe fracture destruction and severe bone pain, severely affecting the quality of life of the patient. The current clinical treatments for bone metastasis include palliative radiotherapy, systemic chemotherapy, osteoclast blocker treatment, analgesic adjuvant treatment, etc. However, all of these are palliative treatments rather than curative treatments. Therefore, there is an urgent need to develop new curative therapies for bone metastasis with few side effects.
Immune checkpoint blocking (Immune checkpoint blockade, ICB) therapy is a cancer immunotherapy that re-activates the immune system by blocking immune checkpoint molecules from attacking tumor cells. Although ICB clinical results appear excellent, many patients with bone metastases are not responsive to ICB. This is due to the characteristics of Tumor Immune Microenvironment (TIME) for bone metastases, high levels of transforming growth factor-beta (TGF-beta) driving CD4 in bone matrix + T cell polarization to T H 17 instead of T H Pedigree 1. At present, TGF-beta is paid attention as a new target point of tumor immunotherapy, and development of related blocking agents is also continuously emerging. In 2019, the combined ghatti and merck combined PD- (L) 1/TGF- β diabody Bintrafusp alfa (M7824) was developed, which was more effective in inhibiting metastasis of bone metastases than TGF- β and PD-L1 mab in multiple preclinical models and clinical trials. However, M7824 caused 21.3% objective remission and 69% immune-related adverse effects in phase 1 trial studies for patients with non-small cell lung cancer (immune-related adverse events, irAEs). Thus, there is a need for further efforts to develop targeted drug delivery systems to reduce the reactivity of irAEs.
Due to the unique transport process of cells, low immunogenicity and high biosafety, more and more researchers have tried to use cells as drug delivery vehicles. In recent decades, erythrocytes, platelets, monocytes, macrophages, stem cells, bacteria and the like have been used to treat cancer, diabetes, pneumonia and the like. Such drug delivery vehicles fall into two main categories: cell-free delivery vehicles (Cell-free delivery vehicles, CFDV) and Cell-based delivery vehicles (Cell-based drug delivery, CBDD). CFDV mainly includes extracellular vesicles, erythrocyte-related vector-based, cell membrane-based nanoparticles, and the like. CFDV does not have critical organelles and loses cell-specific biological activity, so many CFDV often fail to specifically target organs nor respond to chemical signals in the body. CBDD complements this disadvantage and allows to design drug delivery vehicles with specific bioactivity according to the requirements. At present, CBDD has rapidly become a platform for disease treatment, including erythrocytes, inactivated tumor cells, genetically engineered stem cells, immune cells, and the like. Therefore, the invention aims to find a hematopoietic stem cell based on genetic engineering to further improve the therapeutic effect on diseases such as bone metastasis.
Disclosure of Invention
In order to solve the technical problems, the invention discloses a genetically engineered HSCs carrier, and an administration system for bone metastasis therapy is constructed by taking the genetically engineered hematopoietic stem cells as a drug carrier.
It is a first object of the present invention to provide a genetically engineered hematopoietic stem cell drug delivery system comprising hematopoietic stem cells of an immunotherapeutic substance, including but not limited to programmed death receptor 1 (PD-1), T-cell immunoglobulin and mucin domain 3 (T-cell immunoglobulin domain mucin-3, tim-3), lymphocyte Activation Gene 3 (lymphocyte activating gene 3, LAG-3), T cell immunoreceptor with Ig and ITIM domains (T-cell immunoreceptor having Ig and ITIM domains, TIGIT), cytotoxic T lymphocyte-associated antigen-4 (cytotoxic T lymphocyte-associated antigen 4, CTLA-4), anti-HER2 antigen receptor, anti-EGFR antigen receptor, anti-CD3 receptor, anti-F4/80 receptor, CD96, CD80/86, CD40, OX40L, 4-1BBL immunoregulatory molecule, and the like, and a TGF-beta inhibitor loaded on the hematopoietic stem cells.
Furthermore, the hematopoietic stem cells are human, murine, porcine or rabbit hematopoietic stem cells, and are obtained from bone marrow by limiting dilution.
The hematopoietic stem cell (hematopoietic stem cells, HSCs) is a pluripotent stem cell that has all the commonalities of stem cells, namely self-renewal and multipotency. Hematopoietic stem cells are adult stem cells in the blood system, are located in a special hematopoietic microenvironment, are mainly present in bone marrow, and can self-renew and multidirectional differentiate into blood cells with various functions, and maintain the establishment and dynamic balance of the blood system. In order to eliminate ethical problems, the human hematopoietic stem cells are ethically approved human bone marrow hematopoietic stem cells, including commercial human bone marrow hematopoietic stem cells (e.g., human bone marrow cd34+ hematopoietic stem cells of LONZA, etc.).
In particular, the human pluripotent stem cells according to the invention are not capable of developing into an intact individual, and are all pluripotent stem cells that have been established through ethical examination.
Further, the immunotherapeutic is overexpressed by lentiviral transfection, plasmid transfection, or gene editing techniques (e.g., gene insertion), among others. In the invention, a lentiviral vector is adopted for transfection, and a gene pdcd1 encoding PD-1 is connected to the vector and expressed under the regulation of a CMV promoter. For convenient identification, the lentiviral vector is connected with a fluorescent identification marker, and the fluorescent identification marker takes EF1 as a promoter.
Further, the TGF-beta inhibitor is a fat-soluble TGF-beta inhibitor, and the hematopoietic stem cells are loaded with the fat-soluble TGF-beta inhibitor through a phospholipid bilayer.
Further, fat-soluble TGF-beta inhibitors include, but are not limited to SB-505124, galunisertib, TEW-7197, LDN-193189, and the like.
The second object of the present invention is to provide a method for preparing the genetically engineered hematopoietic stem cell drug delivery system, comprising the steps of: the hematopoietic stem cell HSCs-PD-1 over-expressing the immune therapeutic substance is incubated with TGF-beta inhibitor at 1-10 ℃, and the genetically engineered hematopoietic stem cell drug delivery system is obtained by centrifugation.
Further, it is preferred that the cells of the hematopoietic stem cells over-expressing the immunotherapeutic agent are allowed to bind to the TGF-beta inhibitor drug and be released by targeted delivery of the hematopoietic stem cells to the target site, by incubation at 4℃for 4 hours.
Further, the concentration of TGF-beta inhibitor is 30-50 μg/mL.
In the invention, the immune therapeutic substance is firstly over-expressed to enable the immune therapeutic substance to be combined with ligands which are specifically combined with the immune therapeutic substance on tumor cells in focus and cells of marrow system, thereby playing the role of immune checkpoint inhibitor and limiting the development of tumors; secondly, using hematopoietic stem cells as a carrier, over-expressing hematopoietic substancesThe stem cells can be used as a cell to proliferate at a specific part (such as a hematopoietic stem cell bone marrow niche), and further play an anti-tumor role, while antibodies in the prior art (such as PD-L1 antibodies) do not have the proliferation property; finally, the medicine TGF-beta inhibitor is loaded on the hematopoietic stem cells, and the fat medicine TGF-beta inhibitor can be released locally at the focus part to competitively antagonize CD4 in tumor + T cells, promote their orientation to T H 1 and T H Subtype 2 differentiation remodels the local immune microenvironment of the tumor, thereby enhancing the therapeutic effect of immune checkpoint inhibitors.
A third object of the present invention is to provide the use of the above genetically engineered hematopoietic stem cell drug delivery system for the preparation of a medicament for the treatment of bone metastasis.
Further, the administration mode of the medicine is intravenous injection.
In the present invention, bone metastasis refers to tumor bone metastasis, i.e. after tumor cells reach bone marrow along with blood flow, bone tissue is destroyed by interaction with osteoblasts, osteoclasts and bone matrix cells, and various growth factors stored in bone tissue are released, so that tumor cells are continuously proliferated to form metastasis. The tumor is solid tumor such as lung cancer, gastric cancer, colon cancer, melanoma, etc.
In the present invention we depict a cell engineering strategy to replace ICB to target bone using HSCs as a platform. The applicant obtains the HSC with higher purity, and genetically engineer the HSC to make the HSC highly express the immune therapeutic substance, and load and deliver the TGF-beta small molecule inhibitor by using the fat-soluble cell membrane to obtain the genetically engineered HSC carrier loaded with the TGF-beta inhibitor. Hematopoietic stem cells have bone marrow homing effect, which is demonstrated to exhibit powerful bone targeting ability following intravenous injection of genetically engineered HSCs vectors loaded with TGF- β inhibitors. Therefore, the invention is applied to remodelling the immune microenvironment of the bone metastasis tumor, and is used as a living medicine and the activation of T cells at the tumor position and lymph nodes, further research on the capability of improving the tumor immune inhibition microenvironment and blocking the capability of circulating tumor cell exosomes, and finally realize tumor killing and inhibiting the recurrence and metastasis of cancers.
By means of the scheme, the invention has at least the following advantages:
(1) The genetically engineered HSCs loaded with the TGF-beta inhibitor provided by the invention take hematopoietic stem cells as carriers, have the advantages of simple purification and culture, easily available materials, and can replace traditional antibody medicines to play an immune check point role, so that the immune toxicity caused by the antibody of the immune check point inhibitor is reduced, the immunogenicity is low, and the biological safety is good.
(2) The genetically engineered HSCs loaded with the TGF-beta inhibitor can reduce toxic and side effects of the TGF-beta inhibitor and immunological side effects caused by PD-L1 through local targeted therapy. In one aspect, the HSCs-PD-1 released TGF-beta inhibitors competitively bind to CD4 + TGF-beta receptors on T cells promote CD4 + Differentiation of T cells into T H 1 and T H 2 cells, which can reverse the immunosuppressive microenvironment induced by high level TGF-beta in bone metastasis, improving ICB therapeutic effect; on the other hand, the surface of the cell membrane of the HSCs highly expresses PD-1 molecules, can block PD-L1 on tumor and bone marrow cells, and plays an ICB therapeutic role, thereby reactivating the anti-tumor immunity of T cells.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.
FIG. 1 is a graph showing the loading and release of TGF-beta inhibitors for use in the present invention;
FIG. 2 is a graph showing the levels of PD-1 and EGFP expressed by genetically engineered HSCs of the present invention;
FIG. 3 shows the change of key proteins on the front and back surfaces of the small molecule loaded in the genetically engineered cell drug delivery system of the invention;
FIG. 4 shows the levels of PD-L1 and EGFP expressed by genetically engineered melanoma of the present invention
FIG. 5 is the binding capacity of the genetically engineered cell drug delivery system of the invention to melanoma expressing PD-L1 antibodies;
FIG. 6 is a diagram showing the bone homing ability of the genetically engineered cell drug delivery system of the present invention;
FIG. 7 shows the ability of the genetically engineered cell drug delivery system of the present invention to self-replicate in the niche of bone marrow hematopoietic stem cells;
FIG. 8 is a graph showing growth and survival curves of bone metastasis in mice after treatment with the genetically engineered cell drug delivery system of the present invention;
FIG. 9 is a micro-CT image and quantitative analysis of the femur of a mouse after treatment with the genetically engineered cell drug delivery system of the present invention;
FIG. 10 is a graph of H & E and TRAP stained sections of the femur of mice after treatment with the genetically engineered cell drug delivery system of the present invention;
FIG. 11 shows the femoral immune cells (CD 45) of the mice treated by the genetically engineered cell drug delivery system of the present invention + cells) overall change;
FIG. 12 is a graph of femur immunofluorescence staining of mice after treatment with the genetically engineered cell drug delivery system of the present invention;
FIG. 13 shows the change of the femoral immune microenvironment of a mouse after treatment with the genetically engineered cell drug delivery system of the invention;
FIG. 14 is a graph showing tumor distribution imaging and fluorescence intensity quantitative analysis of major organs of mice at day 18 after treatment with the genetically engineered cell drug delivery system of the present invention;
FIG. 15 shows IL-6 levels in mouse serum after treatment with the genetically engineered cell drug delivery system of the invention;
FIG. 16 is a graph of H & E stained sections of other major organs of mice after treatment with the genetically engineered cell drug delivery system of the present invention;
FIG. 17 is a graph showing the simultaneous expression of PD-1 and Tim-3 levels by genetically engineered HSCs of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
C57BL/6 female mice of 6-8 weeks old were purchased from Kwangsi laboratory animal Co. All mouse experiments were performed according to the animal protocol approved by the university of su laboratory animal center.
Mouse melanoma B16F10-luc tumor cells were purchased from Shanghai cell banks. Because the examples are shown in mice, bone marrow-derived Hematopoietic Stem Cells (HSCs) herein were obtained from mice for use in the various embodiments of the invention.
EXAMPLE 1 construction of genetically engineered hematopoietic Stem cell drug delivery platform (SB@HSCs-PD-1)
Configuration of TGF-beta inhibitor solution: a10 mg/mL solution was prepared in DMSO and diluted in PBS to different concentration gradients.
After incubating the TGF-beta inhibitor with the concentration gradient for 4 hours with hematopoietic stem cells, 500g was centrifuged for 3 minutes, and the supernatant was collected and examined for its loading and drug release by high performance liquid chromatography. As shown in FIG. 1, HSCs were incubated with 50 μg/mL TGF- β inhibitors at an encapsulation rate of 14.78+ -2.55%, a loading rate of 5.02+ -0.98 wt.% and a practical level of about 7.39+ -0.49 μg TGF- β inhibition per 10 ten thousand hematopoietic stem cells. TGF- β inhibition released about 77.24 ±4.75% in phosphate buffer at 37 ℃ within 48 hours.
Purified HSCs were incubated with lentiviral vectors (pRLenti-EF 1-EGFP-P2A-Puro-CMV-Pdcd1-3 xFLAG-WPRE) for 48 hours at 37 ℃. Then, 5. Mu.g/mL puromycin was used to screen for HSCs-PD-1 cell lines stably inheriting PD-1 and EGFP, the results are shown in FIG. 2.
SB (50. Mu.g/mL) was incubated with HSCs-PD-1 suspension for 4 hours at 4℃and then centrifuged at 500g for 3 minutes, and the supernatant was discarded to obtain SB@HSCs-PD-1. The results of flow cytometry tests using PerCP-PD-1, PE-Sca-1 and APC-CXCR4 antibodies, as shown in FIG. 3, revealed that the expression of the surface key proteins of HSCs-PD-1 did not show differences before and after loading with SB.
Example 2 relevant characterization of SB@HSCs-PD-1
(1) Binding ability of HSCs-PD-1 to PD-L1 antibodies
Melanoma with high expression of PD-L1 was prepared by transfection of lentivirus (HBLV-m-CD 274-3 xflag-ZsGreen-PURO) and used to detect the PD-L1 binding capacity of HSCs-PD-1, and as a result, as shown in FIG. 4, the modified melanoma cells stably expressed not only ZsGreen signal but also PD-L1. The suspended HSCs-PD-1 and HSCs were co-cultured with adherent PD-L1 expressing melanoma cells B16F10, respectively, for 3 hours, and unbound supernatant cells were removed by washing with PBS. The remaining cells were stained with DAPI and PE-CD44 antibodies, and the results are shown in FIG. 5, which shows that HSCs-PD-1 co-incubation showed a significant increase in CD44 signaling compared to HSCs co-cultured cancer cells, indicating that HSCs-PD-1 can adhere to B16F10-PD-L1 cells through PD-1/PD-L1 binding.
(2) SB@HSCs-PD-1 bone homing ability
HSCs-PD-1 were incubated in DiD with PBS for 30 min to obtain DiD-labeled cells. A portion of the DiD-labeled HSCs-PD-1 was treated with 3.7% paraformaldehyde as a control. DiD-labeled HSCs-PD-1 and paraformaldehyde-fixed DiD-HSCs-PD-1 were intravenously injected into mice. After 4 hours, mice were euthanized and fluorescent imaging by in vivo imaging system, as shown in fig. 6, the bone marrow homing ability of HSCs-PD-1 was significantly stronger than that of formaldehyde-fixed dead cell groups, and SB loading had little effect on the homing ability of HSCs-PD-1.
(3) Self-replication ability of HSCs-PD-1 in the bone marrow hematopoietic stem cell niche
After tail vein injection of HSCs-PD-1, cells in bone marrow and other major organs were harvested on day 0, day 1, day 5, and day 8, respectively. Detection of EGFP Signal on HSCs-PD-1 by flow cytometry, comparison of EGFP in bone marrow + Cell content was varied to assess survival and proliferation of SB@HSCs-PD-1 in vivo. As shown in FIG. 7, HSCs-PD-1 was able to proliferate themselves within the bone marrow hematopoietic stem cell niche, whereas EGFP signal was reduced in other accumulated tissues such as liver and lung, indicating that HSCs-PD-1 were not proliferative in other organs.
EXAMPLE 3 SB@HSCs-PD-1 therapeutic Effect on bone metastasis
The administration is carried out the third day after the establishment of the mouse bone metastasis model,PBS (200. Mu.L) was injected into the tail vein, HSCs (1X 10) 5 Individual cells), SB (7.5 μg), HSC-PD-1 (1X 10) 5 Individual cells), SB@HSCs (1X 10) 5 Individual cells) and SB@HSCs-PD-1 (1X 10) 5 Cells) were injected once every three days for a total of 4 treatments. And bone metastasis size was measured with small animal imaging at specific time points and survival of each group of mice was recorded.
The results are shown in fig. 8, where the bioluminescence signal of bone metastasis was rapidly increased in mice receiving PBS, HSC, SB alone, indicating that bone metastases were not responsive to antibodies to PD-L1. The mice treated with SB@HSCs or HSCs-PD-1 had a slightly reduced burden of bone metastasis compared to control mice, and bone metastasis growth was significantly inhibited following treatment with the genetically engineered hematopoietic cell drug delivery system (SB@HSCs-PD-1). Furthermore, SB@HSCs-PD-1 treatment significantly prolonged survival of mice compared to other groups of mice.
EXAMPLE 4 SB@HSCs-PD-1 inhibiting bone destruction by bone metastasis
(1) micro-CT imaging
After treatment, we randomly selected bone samples from each group and assessed their extent of bone destruction by micro-CT imaging. As shown in fig. 9, the 2D image and 3D reconstruction showed that the trabecular structure was more complete and less damaged in the mice of the sb@hscs-PD-1 treated group compared to the control group. In addition, the bone density (Bone Mass Density, BMD) and bone volume fraction (BV/TV) were significantly higher in the SB@HSCs-PD-1 treated group than in the control group, indicating that SB@HSCs-PD-1 treatment significantly inhibited bone destruction by bone metastases.
(2) H & E and TRAP analysis
The femur of the healthy group, the control group and the SB@HSCs-PD-1 treated group were subjected to pathological sections, and the effect of SB@HSCs-PD-1 on bone destruction was analyzed from the cellular level, as shown in FIG. 10 (the dotted line portion represents the tumor, and the arrowed portion represents the osteoclast). Bone metastases were significantly inhibited following SB@HSCs-PD-1 treatment compared to control. The TRAP staining results showed a decrease in the number of osteoclasts after treatment. These results suggest that SB@HSCs-PD-1 inhibited bone destruction by bone metastases.
Example 5 SB@HSCs-PD-1 pairReverse effect of bone metastasis immunosuppressive microenvironment following treatment with SB@HSCs-PD-1 by cell flow surgery, bone metastasis focus white blood cells (CD 45 + cells) infiltration level was significantly increased (fig. 11). To investigate the recruitment and related activation of the major subtype of immune cells in vivo after treatment. Thus, the effect of SB@HSCs-PD-1 treatment on the immune microenvironment of bone metastases was evaluated by immunofluorescent staining, as shown in FIG. 12. CD4 in bone metastasis in mice receiving SB@HSCs-PD-1 was observed by confocal microscopy compared to untreated mice + And CD8 + Infiltration of T cells increases dramatically.
Flow cytometry analysis was next performed on tumor-infiltrating immune cells collected from bone marrow metastases, as shown in fig. 13. Consistent with confocal results, CD4 was observed in mice treated with SB@HSCs-PD-1 + And CD8 + The proportion of T cells increases significantly, indicating that infiltrating T cells are activated. CD4 + Major subpopulations of T cell infiltrating bone metastasis include TH1, TH2, TH17 and Treg cells. In addition, SB@HSCs-PD-1 treatment increased the proportion of anti-tumor TH1 and TH2 cells, while significantly reducing the proportion of TH17 and Treg cell infiltration. TGF-beta is a regulator of CD4 in bone metastasis microenvironment + T cells like TH17 and an important factor in Treg cell differentiation. We also found that the levels of TGF-beta in bone marrow were significantly reduced following SB@HSCs-PD-1 treatment compared to control. Furthermore, CD45 + And CD11b + CD45 + The level of PD-L1 on cells and tumor cells was significantly reduced following SB@HSCs-PD-1 treatment, indicating that the genetically engineered cell drug delivery system platform of the invention can effectively express PD-1 for a long period of time to block PD-L1.
EXAMPLE 6 Secondary metastatic Effect on bone metastasis following SB@HSCs-PD-1 treatment
Bone metastases are not limited to bone marrow but undergo systemic metastasis with blood, ultimately leading to death of the patient, with lung tissue metastasis being most pronounced. On day 18 post-molding injection, luciferase substrate, euthanasia, organs were collected and analyzed for secondary metastasis of bone metastases by imaging. The results are shown in FIG. 14, which shows that SB@HSCs-PD-1 inhibited not only the growth of bone metastases in mice, but also limited their metastasis to the lungs.
EXAMPLE 7 safety evaluation of SB@HSCs-PD-1
IL-6 levels in mouse serum were detected by the ELSIA kit for IL-6, as shown in FIG. 15. The absence of a significant increase in IL-6 levels in serum of mice following SB@HSCs-PD-1 treatment, compared to controls, indicates that mice are resistant to SB@HSCs-PD-1 treatment. In addition, as in FIG. 16, other major organ H & E stained section scan images of mice also showed no difference from the treatment, demonstrating that SB@HSCs-PD-1 had little toxicity to the mice in treatment.
Similar effects can be achieved by replacing the TGF-beta inhibitor SB-505124 described above with Galunisertib, TEW-7197, LDN-193189, etc.
Example 8 HSCs allow for the simultaneous expression of multiple immune molecules
HSCs were incubated with lentiviral vectors for 48 hours at 37 ℃ by the genetic engineering techniques described above. Then, 5. Mu.g/mL puromycin was used to screen for HSCs cell lines stably inherited PD-1 and TIM-3, the results are shown in FIG. 17.
In summary, the genetically engineered hematopoietic stem cell-based drug delivery vehicle of the present invention competitively binds to CD4 by releasing SB + TGF-beta receptors on T cells promote CD4 + T cells differentiate into TH1 and TH2 cells, reprogramming the local immunosuppressive environment of bone marrow. In addition, therapeutic protein loaded HSCs can block the corresponding ligands on tumor and bone marrow cells, thereby reactivating the anti-tumor responsiveness of T cells. In contrast to inanimate anticancer drugs such as antibody drugs, HSCs loaded with therapeutic proteins act as "live agents" and can self-amplify when entering bone marrow by cell proliferation. They can express therapeutic molecules for long term use in bone metastasis therapy. In addition, side effects and irAEs of other normal organs can be reduced as these "live drugs" have little proliferative capacity in organs other than bone marrow. The technology can also express other therapeutic proteins on HSCs to treat bone related primary diseases. And because of the ease of preparation, plasticity, feasibility and biosafety of HSCs platformsThe new technology may have a broad clinical transformation potential.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (9)
1. A genetically engineered hematopoietic stem cell drug delivery system, characterized by: the genetically engineered hematopoietic stem cell drug delivery system comprises hematopoietic stem cells that overexpress an immunotherapeutic substance, and a TGF- β inhibitor loaded on the hematopoietic stem cells; the immunotherapeutic agent is selected from the group consisting of programmed death receptor 1, and the TGF-beta inhibitor is a fat-soluble TGF-beta inhibitor.
2. The genetically engineered hematopoietic stem cell drug delivery system of claim 1, wherein: the immunotherapeutic agent is overexpressed by lentiviral transfection, plasmid transfection or gene editing means.
3. The genetically engineered hematopoietic stem cell drug delivery system of claim 1, wherein: the hematopoietic stem cells are human, murine, porcine or rabbit hematopoietic stem cells.
4. The genetically engineered hematopoietic stem cell drug delivery system of claim 1, wherein: the fat-soluble TGF-beta inhibitor is selected from one or more of SB-505124, galunisertib, TEW-7197 and LDN-193189.
5. A method for preparing a genetically engineered hematopoietic stem cell drug delivery system as claimed in any one of claims 1-4, comprising the steps of: combining hematopoietic stem cells over-expressing an immunotherapeutic agent with a TGF-beta inhibitor in an amount of 1-10 o Incubating and centrifuging to obtainTo the genetically engineered hematopoietic stem cell delivery system.
6. The method of manufacturing according to claim 5, wherein: the concentration of the TGF-beta inhibitor is 30-50 mug/mL.
7. The method of manufacturing according to claim 5, wherein: the hematopoietic stem cells overexpressing the immunotherapeutic agent are incubated with the TGF- β inhibitor for at least 4 hours.
8. Use of the genetically engineered hematopoietic stem cell drug delivery system of any one of claims 1-4 for the preparation of a medicament for the treatment of bone metastasis.
9. The use according to claim 8, characterized in that: the administration mode is intravenous injection.
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