Antibody-engineered bone marrow stromal cells and uses thereof

Abstract

This invention discloses an antibody-engineered bone marrow stromal cell and its applications, belonging to the field of biomedical technology. Using bone marrow stromal cells as a biological carrier, this invention externally modifies them with immune checkpoint antibodies to form an antibody-coated engineered cell preparation, thereby achieving targeted and precise mitochondrial delivery of exhausted T cells. This cell preparation can be used in combination with other immunotherapies that can induce T cell exhaustion, such as tumor immunotherapies and CAR-T therapy, to help enhance their anti-tumor efficacy and duration of effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody-engineered bone marrow stromal cell and its application in the field of T cell exhaustion. Background Technology

[0002] Sustained low-level TCR stimulation in solid tumors drives T cells into a state of impaired immune response, i.e., T cell exhaustion. The tumor microenvironment is filled with various immunosuppressive factors, including nutrient deprivation, immunosuppressive cytokines, and immunosuppressive cells, all of which affect effector T cell differentiation and induce T cell exhaustion. Exhausted T cells (Tex) are characterized by high expression and often co-expression of inhibitory receptors such as PD-1, TIM-3, CTLA-4, LAG-3, and TIGIT, which significantly weaken the anti-tumor immune effect of T cells, leading to tumor immune escape. T cell exhaustion limits the durability of various immunotherapies, including tumor vaccines and adoptive T cell therapy, and has become a key scientific problem that urgently needs to be solved in the treatment of solid tumors.

[0003] Given the high expression of inhibitory receptors on the surface of Tex cells, scientists have pinned their hopes on immune checkpoint blockade (ICB) therapy, represented by PD-1 antibodies. Although ICB therapy has shown significant efficacy in treating solid tumors and has been shown to alleviate T cell exhaustion to some extent, its overall response rate remains unsatisfactory. A key limiting factor is the heterogeneity of Tex cells; even after blockade with a single antibody, other inhibitory receptors remain highly expressed, mediating immune escape. Subsequent research revealed that T cell exhaustion is not only manifested by the upregulation of external inhibitory receptors but is also accompanied by severe metabolic defects, particularly mitochondrial dysfunction and energy crisis due to decreased mitochondrial quality, constituting the intrinsic characteristics of Tex. Due to the complexity of the causes of T cell exhaustion and the multifaceted nature of Tex's typical characteristics, single therapies often fail to prevent exhaustion differentiation. Therefore, reconstructing T cell function from both internal and external dimensions has become an important means of addressing T cell exhaustion.

[0004] Since a 2006 article published in PNAS first demonstrated that mitochondrial transfer could rescue cells with damaged mitochondria, an increasing number of researchers have focused on exogenous healthy mitochondrial transfer strategies and applied them to the treatment of various diseases. However, direct delivery of live mitochondria faces numerous challenges, such as cumbersome extraction procedures, extremely low yields, difficult storage, and easy loss of viability during extraction. Therefore, indirect mitochondrial delivery using donor cells offers greater advantages. In terms of donor cell selection, bone marrow stromal cells (BMSCs) have attracted considerable attention due to their unique low immunogenicity and high mitochondrial quality. Inspired by this, BMSC mitochondrial transfer strategies hold promise for addressing the problem of T cell exhaustion.

[0005] Current research generally focuses on improving a single characteristic of T cell exhaustion, neglecting the complexity of Tex (therapeutic excitatory function). A single approach often fails to effectively improve T cell effector function in the long term. Among these, multi-combination ICB therapies, which have attracted significant clinical attention, can restore T cell effector function by simultaneously blocking inhibitory receptors on the T cell surface (PD1, CTLA4, LAG3, etc.). Opdullagra, a combination drug of nivolumab (anti-PD1) and renalalimab (anti-LAG3), was launched in 2022 and used to treat metastatic melanoma. However, recent clinical trials (NCT03743766) have shown that while it can achieve some efficacy, T cell exhaustion scores remain high, failing to achieve long-term immune effects.

[0006] Based on this, this invention takes T cell exhaustion as the research object and provides a non-redundant ICB-assisted enhancement strategy, while overcoming the "internal and external troubles" of T cell exhaustion, providing a new adjuvant therapy strategy for clinical tumor vaccines and CAR-T cell therapy. Summary of the Invention

[0007] The main objective of this invention is to address two key characteristics of T cell exhaustion by developing an immune checkpoint antibody-engineered bone marrow stromal cell preparation that overcomes both external immunosuppressive receptors and internal mitochondrial deficiencies. The surface immune checkpoint antibody blocks the highly expressed inhibitory receptors on Tex cells, releasing the "immune brakes." Simultaneously, the antibody targets and anchors Tex cells in vivo, helping to establish a mitochondrial transfer channel between BMSCs and Tex cells. Through the transplantation of healthy mitochondria, the internal mitochondrial quality of Tex cells is restored, thereby compensating for the limitation of ICB therapy in maintaining long-term T cell immune responses.

[0008] A first aspect of the present invention provides antibody-engineered bone marrow stromal cells, comprising bone marrow stromal cells having at least two antibodies modified on their surface; The antibodies are selected from immune checkpoint antibodies, anti-cytokine antibodies, anti-cytokine receptor antibodies, and anti-fibrotic antibodies.

[0009] Furthermore, the immune checkpoint antibody is selected from PD-1 antibody, TIM-3 antibody, CTLA-4 antibody, LAG-3 antibody, and TIGIT antibody.

[0010] Furthermore, the anti-cytokine antibody and anti-cytokine receptor antibody are selected from anti-TNF-α antibody, anti-IL-6 / IL-6R antibody, anti-IL-17 / IL-23 antibody, anti-IL-4 / IL-13 antibody, anti-IL-5 / IL-5R antibody, and anti-GM-CSF antibody.

[0011] Furthermore, the antifibrotic antibody is selected from TGF-β antibody and CTGF antibody.

[0012] Furthermore, two antibodies were modified on the surface of the bone marrow stromal cells.

[0013] Furthermore, the surface of the bone marrow stromal cells is modified with two immune checkpoint antibodies, the mass ratio of the two immune checkpoint antibodies being 10:1 to 1:10, preferably 5:1 to 1:5.

[0014] Furthermore, the antibody is modified on the surface of bone marrow stromal cells in the following manner: (a) Chemical coupling, such as chemical coupling based on amino groups, thiol groups or aldehyde groups on the cell surface; (ii) Bioorthogonal coupling, such as bioorthogonal coupling based on carbohydrate metabolism reactions; (iii) Cell membrane insertion, such as lipid-modified cell membrane insertion; (iv) Non-covalent coupling, such as non-covalent coupling based on biotin-streptavidin.

[0015] Chemical coupling or biological orthogonal coupling is preferred.

[0016] like Figure 1 As shown, in a specific embodiment of the present invention, the antibody-engineered bone marrow stromal cells are prepared using the following steps: Step 1: Bone marrow stromal cells were cultured in MEMα medium containing azide sugar to obtain bone marrow stromal cells expressing azide tags (BMSC-N3).

[0017] The azidosaccharides mentioned therein include, but are not limited to, N-azidoacetylmannosamine (Ac4ManNAz), N-azidoacetylgalactosamine (Ac4GalNAz), and N-azidoacetylglucosamine (Ac4GlcNAz). The concentration of the azidosaccharides is 1-100 µM, preferably 10-50 µM.

[0018] Step 2: Dilute the antibody stock solution in alkaline buffer (preferably 0.5–1 mg / mL), and simultaneously dilute the alkynyl compound -NHS with alkaline buffer to a concentration of 10–100 µM (preferably 20–60 µM). Mix the antibody solution and the alkynyl compound solution in equal volumes and react at 4–25 °C for 1–4 h. Remove the free alkynyl groups by ultrafiltration and centrifugation using an ultrafiltration tube to obtain the alkynyl-conjugated antibody.

[0019] The alkaline buffer solution is PBS with a pH range of 7.5 to 8.5. The alkynyl compound -NHS includes one of dibenzocyclooctyn-NHS, cyclopropanecyclooctyn-NHS, or transcyclooctene-NHS.

[0020] Step 3: In the BMSC-N3 adherent or suspension state, add alkynyl-conjugated antibody at a dosage of 0.5~12µg / 10 5 Cells were reacted at 4°C or 37°C for 0.5–2 h, and then the cells were collected and washed with PBS solution to remove free antibodies, finally obtaining antibody-engineered bone marrow stromal cells.

[0021] The adherent state coupling process is as follows: wash the BMSC-N3 cells in the culture plate / dish with PBS 3-5 times to remove free azide groups, add serum-free MEMα medium containing antibody solution to cover the cells, and then carry out the coupling reaction.

[0022] The suspension coupling process involves: washing and digesting BMSC-N3 to collect it, resuspending it in a centrifuge tube in serum-free MEMα medium containing antibody solution, vortexing to mix, and then performing the coupling reaction.

[0023] A second aspect of the present invention provides the use of the antibody-engineered bone marrow stromal cells described above in the preparation of antitumor products.

[0024] Furthermore, the anti-tumor product can repair T cell depletion.

[0025] Furthermore, the anti-tumor product is a vaccine enhancer or an immunotherapy drug.

[0026] This invention uses bone marrow stromal cells as a biological carrier, externally modified with immune checkpoint antibodies to form antibody-coated engineered cell preparations, thereby achieving targeted delivery to exhausted T cells and precise mitochondrial delivery. The antibody-conjugation efficiency of the antibody-engineered bone marrow stromal cells of this invention is 20%–100%, preferably 60%–90%; the antibody-conjugated amount used for treatment is 2–10 µg / 10 5 For cells, the optimal antibody-conjugated dosage is 2-8 µg / 10g, based on factors such as cell viability and mitochondrial quality after conjugation. 5 cell.

[0027] This invention employs a combination of dual immune checkpoint antibodies, mitigating the risk of tumor immune escape inherent in single-antibody formulations. Furthermore, covalent modification efficiently and specifically anchors the antibodies to the cell surface, enhancing formulation stability. Simultaneously, antibody anchoring increases targeting of exhausted T cells, enabling precise mitochondrial delivery to these cells. This cell preparation can be used in combination with other immunotherapies that induce T cell exhaustion, such as tumor immunotherapies and CAR-T therapy, to enhance their anti-tumor efficacy and duration of effect.

[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a bone marrow stromal cell preparation engineered with dual immune checkpoint antibodies. The dual antibody engineering enhances the targeting of exhausted T cells, and the binding of the antibodies to the inhibitory receptors on the surface of T cells can stabilize the mitochondrial transfer process between cells, promote selective mitochondrial transfer, help exhausted T cells relieve immunosuppression while enhancing mitochondrial metabolic function, thereby reactivating the anti-tumor effect of exhausted T cells.

[0029] 2. This invention provides a method for preparing a modular and replaceable antibody-engineered cell preparation, wherein bone marrow stromal cells can be replaced with other mouse or human bone marrow stromal cells; the immune checkpoint antibodies used can be replaced with antibodies against other biological processes; and the disease model can be replaced from solid tumors to other diseases such as liver and lung fibrosis or myocardial injury.

[0030] 3. The bone marrow stromal cell preparation provided by this invention has the advantages of low MHC molecule expression and low likelihood of inducing immune rejection. It can be rapidly prepared into a ready-to-use cell preparation product for use in allogeneic patients. Furthermore, this cell preparation has been verified to have no tumor-promoting or tumorigenic properties in vivo, demonstrating good biosafety.

[0031] 4. This invention enables the rapid, efficient, and biosafe preparation of an antibody-engineered cell preparation, and has the potential for anti-tumor applications in enhancing vaccines, CAR-T and other immunotherapies. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the preparation process of the antibody-engineered cell preparation in this invention.

[0033] Figure 2 The results are for the cell compatibility of azidosaccharides.

[0034] Figure 3 In the image, A is a flow cytometry image of IgG antibody conjugation, and B is a fluorescence imaging image of BMSC cells with conjugated antibodies.

[0035] Figure 4The effects of cell state (adherent / suspended) and reaction temperature on antibody conjugation efficiency.

[0036] Figure 5 The effect of reaction temperature and reaction time on antibody conjugation.

[0037] Figure 6 The survival rate of BMSC cells under different reaction conditions.

[0038] Figure 7 The effect of antibody engineering on BMSC cell function.

[0039] Figure 8 The results show that antibody engineering enhances the anchoring of exhausted T cells.

[0040] Figure 9 This is a selective result for T cells undergoing mitochondrial transfer.

[0041] Figure 10 It is an antibody-engineered cell preparation to reactivate T cells.

[0042] Figure 11 To enhance the efficacy of antibody-engineered cell preparations for tumor vaccines.

[0043] Figure 12 To ensure the biosafety of cell preparations. Detailed Implementation

[0044] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0047] The BMSCs used in the following examples are immortalized cell lines derived from the skull bone marrow of C57BL / 6 mice, namely OP9 (purchased from Pronosei). Other mouse or human bone marrow stromal cell lines can also be used instead. Example 1

[0048] 1. Preparation of antibody-engineered cell preparations (1) First, the cell compatibility of different concentrations of Ac4ManNaz was investigated using the MTT assay. BMSC cells were seeded in 96-well plates (2×10⁻⁶ cells / wells). 3BMSCs were cultured in MEMα medium supplemented with different concentrations of Ac4ManNaz (0, 5, 10, 25, 40, 50 µM). The medium was changed 1-2 times during the reaction, always replacing it with fresh medium containing Ac4ManNaz. After 72 h, the supernatant was discarded, and MTT solution (0.5 mg / mL) was added. After reacting for 4 h, the supernatant was discarded again, and DMSO was added and shaken for 15 min. The absorbance at 450 nm was then measured using a microplate reader, and the cell viability was calculated using the following formula: .

[0049] Figure 2 The results showed that the survival rate of BMSCs treated with different concentrations of Ac4ManNaz was above 90% in the range of 0-50 µM, demonstrating that the azidosaccharide labeling has good cell compatibility.

[0050] (2) IgG antibody was used as the model antibody. The IgG antibody was dissolved in PBS (pH 8.5) at a concentration of 0.5 mg / mL. The DBCO-NHS (10 mM) solution was diluted with PBS (pH 8.5) to a working solution of 40 µM, and an equal volume of this solution was added to the IgG antibody solution. After thorough mixing, the mixture was reacted at 4°C for 2 h. Then, an ultrafiltration tube (Mw 3 kDa) was used, and the mixture was ultrafiltered and centrifuged three times (4°C, 8000g, 20 min). The liquid remaining in the inner tube was IgG-DBCO. The protein content of the product was determined using the BCA protein quantification method. FITC-NHS and TAMRA-NHS dyes were added to IgG-DBCO, and after thorough mixing, the mixture was reacted at 4°C for 2 h. The mixture was then ultrafiltered and centrifuged using the same method to obtain IgG-DBCO. FITC and IgG-DBCO TAMRA Azide-treated BMSCs were washed 2-3 times with PBS to remove residual Ac4ManNaz and seeded in 24-well plates (4 × 10⁻⁶ cells / well). 4 / well), add IgG-DBCO FITC and IgG-DBCO TAMRA (Based on protein content, 2 µg), a control group was also set up, with unmodified DBCO IgG added. FITC and IgG TAMRA (Based on protein content, 2 µg). After reacting at 37°C for 0.5 h, the cells were washed, collected, and the proportion of positive cells in PE and FITC channels was detected by flow cytometry.

[0051] Figure 3 Image A shows a flow cytometry plot of IgG antibody-conjugated cells, compared with IgG cells without DBCO modification. FITC / TAMRACompared with the control group, DBCO modification significantly improved the antibody labeling rate of BMSC cells, demonstrating the necessity and specificity of the azide-alkynyl click reaction.

[0052] (3) To observe the location of the antibody after the click reaction, azide-treated BMSC cells were seeded in a confocal dish (4 × 10⁻⁶ cells / year). 4 / well), add IgG-DBCO FITC and IgG-DBCO TAMRA (Based on protein content, 2 µg), after reacting at 37℃ for 0.5 h, wash 2-3 times with PBS, add 4% paraformaldehyde solution, fix at room temperature for 15 min, wash 2-3 times with PBS, then add DAPI staining solution for 10 min, and observe and photograph using a laser confocal microscope after washing.

[0053] Figure 3 Image B shows a fluorescence imaging pattern of antibodies conjugated to the surface of BMSC cells, representing IgG antibodies of two different immune checkpoint antibodies. FITC and IgG TAMRA All were labeled on the surface of BMSC cells, with little intracellular distribution, demonstrating the specificity of the click-response modified site.

[0054] 2. Optimization of preparation conditions for antibody-engineered cell preparations (1) Screening and optimization of reaction temperature and cell state in the preparation process: The antibody conjugation efficiency of adherent cells and suspension cells at 4℃ and 37℃ was detected by ELISA to determine the optimal reaction temperature and cell state. For adherent cells, azide-treated BMSCs were seeded in 48-well plates (2×10⁻⁶ cells / wells). 4 / well), after adhesion, add IgG-DBCO (4 µg / 10 wells). 4 Cells were then reacted at 37°C and 4°C for 1 h each. Unbound IgG-DBCO was removed by washing with PBS, and cells were digested and collected. 0.5% Triton X-100 solution was added, and the cells were mixed thoroughly by pipetting. The cells were incubated at 4°C for 1 h, and then centrifuged at 12000 g for 10 min to collect the supernatant. For suspension cells, azide-treated BMSCs were collected in centrifuge tubes, and IgG-DBCO (4 µg / 10000g) was added. 4 Cells were collected, vortexed, and incubated at 4°C and 37°C for 1 h each. Vortexing was repeated midway through the incubation. Cells were washed 2-3 times with PBS and collected by centrifugation. The cells were then processed again using the same procedure, and the supernatant was diluted 100-fold. All samples were analyzed using an ELISA kit, and the antibody-conjugation efficiency for each sample was calculated using the following formula:

[0055] .

[0056] Figure 4 To investigate the antibody conjugation efficiency of BMSCs in different states at different reaction temperatures, the conjugation efficiency of BMSCs in adherent state after 1 h of reaction was generally lower than that in suspension state. The antibody conjugation efficiency of BMSCs in suspension state after 1 h of reaction at 4℃ was as high as 75%, which was higher than the conjugation efficiency at 37℃ (only 40%). Therefore, suspension state and reaction at 4℃ are preferred.

[0057] (2) Screening and optimizing the reaction time of the preparation process: Collect azide-treated BMSCs in centrifuge tubes and add IgG-DBCO TAMRA (4 µg / 10 4 Cells were reacted at 37℃ and 4℃ for 0.5 h, 1 h, and 2 h, respectively. Cells were then collected and washed 2-3 times with PBS. The proportion of PE-positive cells was detected by flow cytometry; these were IgG-conjugated BMSCs. Simultaneously, the activity of BMSCs reacted at 4℃ and 37℃ for 0.5 h, 1 h, and 2 h was assessed using the MTT assay, with the specific experimental methods described above. Finally, the optimal conditions were selected by comprehensively considering cell activity, efficiency, and reaction rate.

[0058] Figure 5 The proportion of antibody-conjugated BMSCs was measured at 4℃ and 37℃ for different reaction times. At 4℃, the conjugation rate increased with increasing time, with no significant difference between 1 h and 2 h. At 37℃, the conjugation rate decreased with increasing reaction time. Therefore, the preferred reaction temperature is 4℃ and the reaction time is 1 h.

[0059] Figure 6 The survival rates of antibody-conjugated BMSCs were measured at 4℃ and 37℃ for different reaction times. Cell survival rates at 4℃ were generally higher than 90%, while survival rates at 37℃ decreased with increasing reaction time. Based on these results, the optimal reaction temperature was 4℃.

[0060] 3. Effects of antibody conjugation on BMSC cells (1) To investigate the effect of antibody conjugation on BMSC cell function, cell viability and cell membrane integrity were first examined: the MTT assay was used to investigate the cytotoxicity of different antibody dosages at the following levels: 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, and 1 µg / 5000 cells. The specific method was the same as described above, and the cell viability of BMSCs was calculated. In addition, azide-conjugated BMSCs were seeded in 96-well plates (5 × 10⁶ cells / well). 3After cell adhesion, IgG-DBCO (0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1 µg / 5000 cells) was added to each well and incubated at 37°C for 0.5 h. The amount of lactate dehydrogenase released by the cells was measured according to the procedure of the lactate dehydrogenase assay kit (Beyotime) to assess the integrity of the cell membrane.

[0061] Figure 7 In Figure A, the survival rate of BMSC cells with different antibody addition amounts is shown. Within the range of 0~1 µg / 5000 cells, antibody conjugation does not significantly reduce cell viability and has good safety.

[0062] Figure 7 In the figure, B represents the lactate dehydrogenase release rate. Within the antibody mass range of 0~1 µg / 5000 cells, the lactate dehydrogenase release rate is less than 2%, indicating that antibody conjugation does not cause cell membrane damage.

[0063] (2) ATP production detection: ATP production is a direct indicator of mitochondrial metabolic activity. Using an ATP detection kit (Beyotime), the concentration of ATP produced by cells after antibody conjugation was detected as described above.

[0064] Figure 7 C represents the intracellular ATP concentration, and the results indicate that 0-4 µg / 10 5 Within the range of antibody-conjugated amounts, the ATP production capacity of BMSC cells did not decrease significantly.

[0065] Detection of key mitochondrial translocation protein: RHOT1 is a key protein regulating mitochondrial translocation via TNT and is responsible for driving mitochondrial transport. Intracellular RHOT1 expression after antibody conjugation was detected by Western blot. In short, azide-treated BMSCs were seeded in 6-well plates (1×10⁻⁶ cells / wells). 5 / well), incubate overnight. Add IgG-DBCO (0, 0.5, 1, 2, 4, 8, 12 µg / 10 wells). 5Cells were incubated at 37°C for 0.5 h, washed 2-3 times with PBS, and cultured in fresh medium for another 24 h. Cells were then lysed on ice, scraped and collected, and subjected to low-temperature sonication for 1 min (3 s on, 6 s off). Protein was extracted by centrifugation at 12000 g for 20 min. Protein quantification was performed using a BCA kit. The remaining protein sample was added to 5× loading buffer, boiled for 5 min, and then subjected to SDS-PAGE electrophoresis, membrane transfer, and blocking. The sample was incubated overnight at 4°C with primary antibodies RHOT1 (1:1000 dilution) and GAPDH (1:3000 dilution). The following day, the sample was incubated at room temperature with HPR-labeled secondary antibody (1:5000 dilution) for 1 h. After washing, the gel was developed using a gel imaging system, and the band gray values ​​were semi-quantitatively analyzed using ImageJ.

[0066] Figure 7 DE represents the RHOT1 expression in cell formulations with different antibody-conjugated amounts, indicating that 0-4 µg / 10 5 Within the range of antibody-conjugated amounts, RHOT1 expression in BMSC cells was not significantly affected. Therefore, the preferred antibody-conjugated amount is 4 µg / 10 5 cell. Example 2

[0067] Investigation of T cell anchorage after antibody conjugation: BMSC cells and azide-treated BMSC cells were seeded in confocal dishes (4 × 10⁻⁶ cells / year). 4 / well), after cells adhered, they were labeled with Cell Trace Far Red, washed, and then aPD1-DBCO and aLAG3-DBCO (mass ratio 3:1, 4 µg / 10 wells) were added. 5 Cells), reacted at 37°C for 0.5 h. Then, 2 × 10⁻⁶ pre-stained CFSE-exhausted T cells were added. 5 ( / well) Continue co-culturing for 24 h. Wash 2-3 times with PBS to remove free Tex cells, then fix with 4% paraformaldehyde at room temperature for 15 min, then add DAPI staining for 10 min, wash with PBS 2-3 times, observe the adhered T cells using laser confocal microscopy, and analyze the counting using Image J.

[0068] Figure 8 Laser confocal images and T cell count analysis of AB-anchored T cells were performed, where green fluorescence represents T cells and red fluorescence represents BMSCs or antibody-engineered BMSCs (eBMSCs). The results showed that more T cells adhered and remained in the eBMSC group, indicating that antibody conjugation can increase T cell anchoring and may enhance the probability of mitochondrial translocation. Example 3

[0069] T-cell selectivity for mitochondrial transfer: A 3D tumor sphere model was constructed to investigate whether antibody-engineered eBMSCs exhibit selectivity for the transfer of both T cells and tumor cells. LLC cells were uniformly seeded in 96-well U-shaped plates (3000 cells / well) and cultured statically for 5 days. The culture medium was then changed, and pre-stained MitoTracker Red CMXRos-containing BMSCs / eBMSCs and pre-stained CFSE-containing T cells were added separately (BMSC / eBMSC:T cell ratio 1:1). After co-culturing for 16 h, the tumor spheres were transferred to new wells for soaking and washing, followed by fixation with 4% paraformaldehyde for 15 min and nucleus labeling with DAPI. After washing again, the fluorescence distribution of the tumor spheres was scanned layer by layer using the Z-stack function of a laser confocal microscope.

[0070] Figure 9 The images show laser confocal tomographic images of tumor spheres, with red representing mitochondrial fluorescence from BMSCs or eBMSCs and green representing T cells. The results show that red mitochondrial fluorescence in the BMSC group is widespread throughout the tumor spheres and not limited to T cells; while mitochondrial fluorescence in the eBMSC group is significantly co-localized with the green fluorescence of T cells, indicating that antibody conjugation can enhance the selective mitochondrial transfer of cell preparations to T cells. Example 4

[0071] (1) ATP production capacity of depleted T cells after treatment: aPD1 / aLAG3 engineered eBMSCs were prepared using the same method as described above and co-cultured with Tex cells for 24 h. Separate antibody groups (with a dose equivalent to the eBMSC conjugation amount) and unmodified BMSC cell groups were also established. Tex cells in the supernatant were collected, and the ATP concentration produced within the Tex cells after different treatments was detected according to the steps of the ATP detection kit.

[0072] Figure 10 In the figure, A represents the intracellular ATP concentration of exhausted T cells. The results showed that compared with immune checkpoint antibody therapy alone, eBMSC treatment significantly enhanced the ATP production capacity of exhausted T cells, indicating improved mitochondrial activity.

[0073] (2) Exhaustion indicators of exhausted T cells after treatment: aPD1 / aLAG3 engineered eBMSCs were prepared using the same method as above, and the same grouping was set. Exhausted T cells were added and co-cultured for 24 h. The expression ratio of PD1 and LAG3 in exhausted T cells after different treatments was detected by flow cytometry.

[0074] Figure 10The B-value represents the double positivity of PD1 and LAG3 on the surface of exhausted T cells after different treatments. High expression and co-expression of PD1 and LAG3 are important indicators of exhaustion. Compared with immune checkpoint blockade alone, PD1 expression was significantly higher after eBMSC treatment. + LAG3 + The proportion of T cells decreased further, indicating that the cell preparation has a better effect on improving T cell exhaustion. Example 5

[0075] Antitumor efficacy study of eBMSC adjuvant vaccine: First, OVA protein vaccine was prepared (50 mg of 4-carboxyphenylboronic acid, 115 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 69 mg of N-hydroxysuccinimide (NHS) were accurately weighed, dissolved in a mixed solvent (ethanol:water = 1:1), and stirred at room temperature for 1 h to activate the carboxyl groups on 4-carboxyphenylboronic acid. 100 mg of PEI was added, and the reaction was continued at room temperature for 24 h. The reaction solution was dialyzed in double-distilled water for 48 h, with the double-distilled water being changed every 4 h. The dialyzed reaction solution was lyophilized to obtain the product PP. 5 mg of PP was dissolved in 20 mM HEPES buffer to a concentration of 1 mg / mL; 5 mg of OVA protein was dissolved in the same buffer to a concentration of 1 mg / mL. The two were mixed in equal volumes and reacted for 0.5 h to obtain a solution with an opalescent appearance for later use.) Next, an orthotopic lung cancer model was constructed in C57BL / 6J mice. LLC-luc-OVA cells were digested and collected, mixed 1:1 with matrix gel, and injected slowly (5×10⁻⁶ cells) into the lung lobe between the 3rd and 4th ribs on the left side of the mouse using an insulin needle. 5 After pausing for 1 minute, the needle was withdrawn and sutured. Mice were randomly divided into 4 groups and administered Saline, Vaccine (OVA protein vaccine, OVA dose 50 µg / mouse), and eBMSC (1×10⁻⁶ mcg / mouse), respectively. 6 / animal, antibody conjugation amount is 4 µg / 10 5 Mice were treated with cells (eBMSCs) and eBMSCs + Vaccine (dose as above). Body weight was continuously monitored during treatment. One week after treatment, some mice were sacrificed, and lung tumor tissue was collected, photographed, and weighed. The remaining mice were monitored for survival.

[0076] Figure 11Figure A shows a schematic diagram of the animal experiment process; Figure B shows the weight monitoring of mice. Except for the saline group, the weight of mice in other groups did not decrease significantly, indicating that the preparation has good safety; Figures C and D show the dorsal side view of lung tumor tissue and tissue quality. It is clear that the eBMSC+Vaccine group has the best tumor inhibition effect; Figure E shows the survival time of mice with cancer. The results show that the eBMSC+Vaccine group can significantly prolong the survival time of mice with lung cancer, and two of them were cured. Example 6

[0077] (1) Investigation of the tumorigenicity and tumorigenicity of bone marrow stromal cell preparations: A subcutaneous 4T1 breast cancer model was constructed in BALB / c mice, and 1×10⁻⁶ mc² of bone marrow stromal cell preparations were subcutaneously injected into the tumor site. 6 One BMSC per mouse (injected twice every 3 days) was used to record changes in tumor volume and compare the tumor growth with that of untreated control mice to evaluate whether BMSCs can promote tumor growth.

[0078] Tumorigenicity: To investigate whether the BMSC cell-derived preparation could spontaneously induce tumors, immunodeficient nude mice were selected as animal models. Subcutaneous injections of 5 × 10⁶ cells were administered. 6 One BMSC cell or 1×10 6 We collected LLC lung cancer cells and used healthy mice as controls to monitor changes at the injection site and tumor growth over a long period of time, and recorded the data with photographs.

[0079] Figure 12 Image A shows the tumor volume change in breast cancer mice, indicating that BMSCs do not have tumor-promoting properties. Image B shows the changes at the injection site in nude mice. Compared with the tumorigenic LLC cell line, BMSCs did not form tumor-like tissue after inoculation, indicating that cell preparations derived from BMSCs are not tumorigenic.

[0080] (2) In vivo tissue and organ compatibility study: Healthy C57BL / 6J mice were injected via tail vein with Antibody (aPD1:aLAG3 ratio of 3:1, total dose of 2 mg / kg) and eBMSC (1×10⁻⁶ mg / kg). 6 / animal, antibody conjugation amount is 4 µg / 10 5 (Cells), administered once every three days. After 14 days, the mice were sacrificed, and blood was collected from the heart, liver, spleen, lungs, and kidneys for routine blood tests, blood biochemistry tests, and H&E staining analysis.

[0081] Figure 12(CL) represents the levels of red blood cell count, hemoglobin content, hematocrit, mean corpuscular volume, platelet count, white blood cell count, blood urea nitrogen, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin after drug administration; (M) represents H&E stained sections of major organs. The comparison shows that the eBMSC antibody-engineered cell preparation provided by this invention does not cause significant damage to the major organs, blood cells, or liver and kidney metabolic functions in mice, indicating its good biocompatibility.

Claims

1. An antibody-engineered bone marrow stromal cell, characterized in that, Including bone marrow stromal cells, the surface of which is modified with at least two antibodies; The antibodies are selected from immune checkpoint antibodies, anti-cytokine antibodies, anti-cytokine receptor antibodies, and anti-fibrotic antibodies.

2. The bone marrow stromal cells according to claim 1, characterized in that, The immune checkpoint antibodies are selected from PD-1 antibody, TIM-3 antibody, CTLA-4 antibody, LAG-3 antibody, and TIGIT antibody.

3. The bone marrow stromal cells according to claim 1, characterized in that, The anti-cytokine antibodies and anti-cytokine receptor antibodies are selected from anti-TNF-α antibodies, anti-IL-6 / IL-6R antibodies, anti-IL-17 / IL-23 antibodies, anti-IL-4 / IL-13 antibodies, anti-IL-5 / IL-5R antibodies, and anti-GM-CSF antibodies.

4. The bone marrow stromal cells according to claim 1, characterized in that, The antifibrotic antibody is selected from TGF-β antibody and CTGF antibody.

5. The bone marrow stromal cells according to claim 1, characterized in that, Two types of antibodies were modified on the surface of the bone marrow stromal cells.

6. The bone marrow stromal cells according to claim 5, characterized in that, Two immune checkpoint antibodies are modified on the surface of the bone marrow stromal cells, with a mass ratio of 10:1 to 1:10, preferably 5:1 to 1:

5.

7. The bone marrow stromal cells according to any one of claims 1-6, characterized in that, The antibody is modified on the surface of bone marrow stromal cells in the following manner: (a) Chemical coupling; (ii) Biological orthogonal coupling; (iii) Cell membrane insertion; (iv) Non-covalent coupling.

8. The use of antibody-engineered bone marrow stromal cells according to any one of claims 1-7 in the preparation of antitumor products.

9. The application according to claim 8, characterized in that, The anti-tumor product repairs T cell depletion.

10. The application according to claim 8, characterized in that, The anti-tumor product is a vaccine enhancer or an immunotherapy drug.

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