Delivery scaffolds for therapeutic cells and methods of making the same

Abstract

The application discloses a delivery scaffold of therapeutic cells and a preparation method thereof. The preparation method comprises the following steps: freezing and drying a lymph node, and obtaining the delivery scaffold. In the application, the lymph node is used as the delivery scaffold, and has good biocompatibility, and has the advantages of keeping CAR-T cell activity and better proliferation of the CAR-T cells; the lymph node loaded with the CAR-T cells can effectively inhibit the growth of tumors.

Description

Technical Field

[0001] This invention relates to the field of drug delivery, and more specifically to a therapeutic cell delivery scaffold and its preparation method. Background Technology

[0002] Surgical resection is the primary treatment for many solid tumors, but residual tumor cells and circulating tumor cells can still induce tumor recurrence. Therefore, chemotherapy and radiotherapy are often used to prevent recurrence of residual tumor cells and metastases, but these therapies are often accompanied by severe toxic side effects.

[0003] Compared to chemotherapy and radiotherapy, which indiscriminately attack both tumor and normal cells, causing severe toxic side effects, novel immunotherapies can train the immune system to specifically recognize and kill tumor cells, thus reducing these side effects. Among them, adoptive cell therapy, represented by chimeric antigen receptor T-cell (CAR-T cell) therapy, has shown remarkable efficacy in treating hematological malignancies.

[0004] However, the immunosuppressive microenvironment within solid tumors limits the survival and proliferation of T cells at the tumor site. Furthermore, insufficient infiltration of intravenously infused CAR-T cells into solid tumors severely restricts the efficacy of CAR-T cell therapy for solid tumors. Therefore, increasing research focuses on developing technologies for in situ delivery of CAR-T cells to solid tumors to maintain their viability in the tumor microenvironment, promote proliferation, and enhance infiltration. These technologies include implantable or injectable gels, microneedles, and scaffolds. Since these materials are all artificially synthesized or designed, although most contain cytokines such as interleukin-2, 7, or 15 to maintain T cell activity, they cannot completely and realistically mimic the support provided to T cells by natural lymphoid organs.

[0005] Since the aforementioned artificial materials used for in situ delivery of solid tumors cannot adequately support T cells, there is an urgent market need for an in situ delivery product for solid tumors that can improve support for T cells.

[0006] Lymph nodes are the sites supporting the proliferation and differentiation of T cells and B cells. Their stromal environment provides excellent support for T cells, and the combination of various cytokines naturally present in lymph nodes is optimal for T cell development. Clinically, lymph node dissection is performed during tumor resection to remove lymph nodes that may have metastasized, thereby reducing the risk of tumor recurrence. These lymph nodes can be utilized as natural scaffold materials to promote the survival and proliferation of CAR-T cells, enabling the localized, sustained delivery of CAR-T cells to the lesion site. Summary of the Invention

[0007] To address the shortcomings of existing artificially synthesized or designed products for in situ delivery of solid tumors, which fail to adequately support T cells, this invention provides a therapeutic cell delivery scaffold and its preparation method. In this invention, lymph nodes, used as delivery scaffolds, maintain good biocompatibility, can be internalized in vivo, and possess the advantages of maintaining CAR-T cell activity and promoting better CAR-T cell proliferation; lymph nodes loaded with CAR-T cells can effectively inhibit tumor growth.

[0008] The first aspect of the present invention provides a method for preparing a delivery stent, wherein the preparation method includes the following steps: freezing and drying lymph nodes to obtain the delivery stent.

[0009] In some embodiments, the preparation method includes one or more of the following conditions:

[0010] The lymph nodes are human lymph nodes;

[0011] The lymph nodes are lymph nodes that do not contain metastatic tumor cells;

[0012] The freezing is achieved by placing the lymph nodes at -30 to -80°C or in liquid nitrogen. For example, the -30 to -80°C can be achieved by placing the lymph nodes in a -80°C freezer or dry ice.

[0013] The drying process is freeze-drying.

[0014] In some implementations, the freezing time is 2 min to 240 min.

[0015] In some implementations, the drying time is 4 to 20 hours.

[0016] In some embodiments, the preparation method further includes the step of cleaning the lymph nodes.

[0017] In some implementations, the washing is performed in a pre-cooled PBS solution.

[0018] The second aspect of the present invention provides a delivery stent obtained by the preparation method described in the first aspect of the present invention.

[0019] A third aspect of the present invention provides a delivery scaffold-cell complex comprising the delivery scaffold described in the second aspect of the present invention, and cells for therapeutic use.

[0020] In some implementations, the cells used for treatment include engineered immune cells and stem cells.

[0021] In some embodiments, the engineered immune cells are selected from CAR-T cells, TCR-T cells, CAR-NK cells, and CAR-M cells; for example, the engineered immune cells are CAR-T cells.

[0022] In some embodiments, the delivery scaffold-cell complex is prepared by injecting cells for treatment into the delivery scaffold.

[0023] In some embodiments, the concentration-to-mass ratio of the cells used for treatment to the delivery scaffold is (2 × 10⁻⁶). 6 (each) / g.

[0024] In some embodiments, the cells used for treatment are centrifuged or precipitated and then resuspended in FBS-free X-Vivo medium or PBS, the suspension is drawn up using a syringe and injected into the delivery scaffold.

[0025] In some implementations, the resuspension is performed on ice.

[0026] In some implementations, the injection is performed on ice.

[0027] In some embodiments, when the cells used for treatment are CAR-T cells, the amino acid sequence of the CAR molecule is as shown in SEQ ID NO:2.

[0028] In some embodiments, the nucleotide sequence encoding the CAR molecule is shown in SEQ ID NO:1.

[0029] The fourth aspect of the present invention provides the use of the delivery scaffold-cell complex described in the third aspect of the present invention in the preparation of a medicament for inhibiting tumors.

[0030] In some implementations, the tumor is selected from gastric cancer, colorectal cancer, pancreatic cancer, cervical cancer, lung cancer, and prostate cancer.

[0031] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0032] The reagents and raw materials used in this invention are all commercially available.

[0033] The positive and progressive effects of this invention are as follows:

[0034] (1) Lymph nodes, as delivery scaffolds, maintain good biocompatibility and have the advantages of maintaining CAR-T cell activity and enabling CAR-T cells to proliferate better.

[0035] (2) Lymph nodes carrying CAR-T cells can effectively inhibit tumor growth. Attached Figure Description

[0036] Figure 1A This is a cryo-electron micrograph of a lymph node after freeze-drying. The scale bar is 10 micrometers.

[0037] Figure 1B This is a cryo-electron micrograph of a lymph node that has been freeze-dried and then loaded with CAR-T cells. The scale bar is 10 micrometers, and the arrows indicate CAR-T cells.

[0038] Figure 2 This diagram illustrates the content of various cytokines in lymph nodes before and after freeze-drying.

[0039] Figure 3 This is a schematic diagram of the in vitro release of CAR-T cell-loaded lymph nodes after lyophilization.

[0040] Figure 4A This is a staining image of live and dead CAR-T cells released from lymph nodes after lyophilization.

[0041] Figure 4B This is a schematic diagram of CCK-8 cell viability test data.

[0042] Figure 5 Hematoxylin and eosin staining of the tissue of a freeze-dried lymph node after subcutaneous implantation in a mouse.

[0043] Figure 6A A flow cytometry representation of CFSE staining of CAR-T cells in a lymph node containing CAR-T cells.

[0044] Figure 6B A statistical graph of the fluorescence intensity of CFSE staining in CAR-T cells within a lymph node containing CAR-T cells.

[0045] Figure 6C A schematic diagram showing the absolute number of CAR-T cells labeled on counting microspheres within a lymph node containing CAR-T cells.

[0046] Figure 7 This is a schematic diagram showing the comparison of cell proliferation promotion between the freeze-dried lymph node group and the artificial material + cytokine group.

[0047] Figure 8A Representative bioluminescence images of tumors in mice at different days after treatment in each group.

[0048] Figure 8B This is a schematic diagram showing the changes in tumor bioluminescence intensity in mice of different groups after surgery as the number of days of treatment increased.

[0049] Figure 8C This is a statistical chart showing the tumor quality of mice in each group 24 days after surgery.

[0050] Figure 8D The survival curves of mice in each group 24 days after surgery are shown.

[0051] Figure 8E This diagram illustrates the levels of tumor cytokines IFN-γ and IL-2 in each group.

[0052] Figure 9A Tumor growth curves for each group in a patient-derived tumor recurrence model.

[0053] Figure 9B Images of tumors in mice from different groups after 30 days of treatment in a patient-derived tumor recurrence model. Detailed Implementation

[0054] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0055] Example 1: Preparation of freeze-dried lymph nodes and loading of CAR-T cells

[0056] Clinically obtained lymph nodes are first placed in sterile, ice-cold PBS solution to remove blood and residual tissue, then the lymph nodes are peeled off and cleaned. They are then frozen at -80 degrees Celsius or directly immersed in liquid nitrogen for 3–4 hours. Once the tissue is completely frozen solid, it is transferred to a freeze dryer and freeze-dried for 4–20 hours to obtain the final product.

[0057] The lymph nodes were obtained from lymph node dissection performed on cancer patients during tumor resection surgery. Pathological examination confirmed that they did not contain metastatic tumor cells.

[0058] The CAR-T cells to be loaded are centrifuged into a pellet, resuspended and concentrated in 10-20 μL of FBS-free X-Vivo medium or PBS. The suspension is then drawn up using an insulin syringe and injected into lyophilized lymph nodes. The resuscitation and injection are performed on ice.

[0059] After lymph nodes are freeze-dried, pores or cavities will form inside the lymph nodes. Figure 1A These pores or cavities can be used to load CAR-T cells. Figure 1B ).

[0060] Example 2: Detection of cytokine content in lymph nodes before and after freeze-drying

[0061] Fresh lymph nodes and an equal amount of lyophilized lymph nodes were cut into pieces and added to RIPA strong lysis buffer (Merck, 20-188) containing 1 mM PMSF, and lysed on ice. The levels of IL-2 (70-EK102-96), IL-7 (70-EK107-96), IL-12p70 (70-EK112-96), IL-15 (70-EK115-96), IL-21 (70-EK121-96), and IFN-γ (70-EK180-96) in the lysis buffer were then detected using an ELISA kit (Linke Biotech). Protein content was determined based on tissue quality for each group.

[0062] The results showed that the cytokine production of freeze-dried lymph nodes was not reduced compared to that of unfreezed lymph nodes. Figure 2 This indicates that freeze-dried lymph nodes contain abundant and diverse cytokines, which play a crucial role in maintaining the survival of CAR-T cells and promoting their effective proliferation. Therefore, freeze-dried lymph nodes have a stronger ability to deliver CAR-T cells.

[0063] Example 3: In vitro release of CAR-T cells from lymph nodes and assay of cell viability

[0064] 4×10 5 CAR-T cells were loaded into lyophilized lymph nodes and immersed in X-Vivo medium without growth factors. The number of free CAR-T cells in the medium was counted at 1, 2, 4, 8, 24, 36, 48, and 96 hours. The results showed that the number of released CAR-T cells gradually increased with time, reaching over 3.5 × 10⁻⁶ after 96 hours. 5 indivual( Figure 3 This demonstrates a strong ability to release cells. Therefore, using lyophilized lymph nodes for in-situ delivery of CAR-T cells can also achieve sustained release at the administration site.

[0065] The present invention also performs live / dead staining and CCK-8 cell viability testing on the released cells.

[0066] First, a hyaluronic acid hydrogel synthesized from artificial materials is prepared as follows:

[0067] Preparation method of hyaluronic acid hydrogel: Dissolve 2.0 g of hyaluronic acid (Bloomage Biotech) in 100 mL of distilled water and stir overnight at 4 °C. Then, add 1.6 mL of methacrylic anhydride dropwise to the HA solution. Add 5 N sodium hydroxide solution to maintain the pH at 8.0–9.0, and then stir continuously at 4 °C for 24 hours. Afterward, pour the entire reaction solution into anhydrous ethanol in batches to precipitate m-HA, filter, and wash three times with anhydrous ethanol. Finally, dissolve the precipitated m-HA with distilled water and dialyze in distilled water for 48 hours. After lyophilization, purified m-HA is obtained. Mix 400 μL of m-HA aqueous solution (4% w / v) with MBA (MBA:m-HA, 1:5, w:w) thoroughly, and add photoinitiator Irgacure 2959 (0.1% w:v). The solution is dissolved using an ultrasonicator. The solution was then added to a 48-well plate and irradiated with UV light for 25 minutes to obtain a hydrogel. The prepared hydrogel was then frozen at -80°C and freeze-dried for 48 hours to obtain hyaluronic acid hydrogel.

[0068] A hydrogel loaded with an equal volume of CAR-T cells (with added cytokines) was subjected to cell release from a lyophilized lymph node loaded with CAR-T cells for a period of time. On the third day, the CAR-T cells released from the lymph node were stained with calcein and PI dyes. Confocal microscopy revealed that the vast majority of cells were viable. Figure 4A This indicates that CAR-T cells can survive better in freeze-dried lymph nodes.

[0069] Furthermore, CCK-8 assays revealed that CAR-T cells within lymph nodes maintained good activity for up to two weeks, while CAR-T cells released via in situ delivery using hyaluronic acid hydrogels showed a decreasing trend in activity. Figure 4B This demonstrates that freeze-dried lymph nodes provide better support for T cell activity, meaning that freeze-dried lymph nodes maintain good biocompatibility and have the advantage of preserving CAR-T cell activity.

[0070] Example 4: Biocompatibility test of freeze-dried lymph nodes in mice

[0071] Lymph nodes were harvested from healthy 6-8 week old C57BL / 6 mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.), freeze-dried, and then re-implanted subcutaneously. Lymph nodes and surrounding skin tissue were removed on days 1, 3, and 7. Surgical suturing and hyaluronic acid gel implantation served as control groups. Inflammation in the surrounding tissue caused by lymph node implantation began to subside on day 3, with only minor inflammation remaining on day 7. The inflammation level was comparable to that of the control group treated with surgical suturing alone. However, hyaluronic acid gel implantation resulted in significant inflammatory response and the formation of fibrous capsules throughout all seven days. This indicates that freeze-dried lymph nodes have good biocompatibility.

[0072] Example 5: Measurement of CAR-T cell proliferation

[0073] 1×10 6 CAR-T cells were loaded into hydrogels containing IL-7 and IL-15 and lyophilized lymph nodes, respectively. After three days of incubation, the free CAR-T cells were harvested. Cells were fixed with 4% paraformaldehyde, centrifuged at room temperature for 15 minutes to remove paraformaldehyde, and washed three times with PBS containing 1% BSA. True-Nuclear... TM Cell transmembrane insertion was performed using a Transcription Factor Buffer Set (424401) (Biolegend, USA). The recommended amount of Ki-67 flow cytometry antibody (Biolegend, USA, 350506) was added for staining for 30 minutes. After washing three times with PBS solution containing 1% BSA, the cells were analyzed by flow cytometry.

[0074] CAR-T cells and a hydrogel control group supplemented with IL-7 and IL-15 were stained with CFSE dye (Biolegend, 423801, USA). The results showed that, compared with the control group, the CFSE fluorescence intensity of CAR-T cells in lyophilized lymph nodes (i.e., the lymph node group) was significantly decreased. Figure 6B Furthermore, both the flow cytometry results from CFSE staining and the absolute cell count results from microsphere counting indicated that the number of CAR-T cells in the lymph node group was higher than that in the control group. Figure 6A and Figure 6C This indicates that CAR-T cells within lymph nodes can proliferate and grow more rapidly, meaning that lymph nodes can effectively promote CAR-T cell proliferation. Compared to the hydrogel group supplemented with IL-7 and IL-15, the Ki-67 index of CAR-T cells cultured in lyophilized lymph nodes was significantly increased. Figure 7 This indicates that CAR-T cells have significantly proliferated.

[0075] Example 6: In vivo efficacy experiment

[0076] Cell line-derived tumor recurrence models:

[0077] One hundred and twelve healthy female NSG immunodeficient mice (Southern Model Biotechnology Co., Ltd.) aged 6–8 weeks were used. After the tumors in the NSG mice, derived from the HeLa cervical cancer cell line, grew to 150 mm³, they were surgically removed, leaving 5% of the tumor intact to simulate postoperative recurrence. The mice were then treated with PBS, peritumoral injection, factor-loaded gel, CAR-T cell-loaded gel, CAR-T cell and cytokine-loaded gel, lymph nodes, and CAR-T cell-loaded lymph nodes. Among the different treatment groups, the CAR-T cell-loaded lymph node group showed the best effect in inhibiting postoperative recurrence and exhibited the slowest tumor growth. Figure 8A and Figure 8B While the peritumoral injection group and the experimental group implanted with CAR-T cell-loaded hydrogel showed tumor inhibition, their inhibition efficiency was lower than that of the CAR-T cell-loaded lymph node group. Tumor mass was measured in the different treatment groups, and the results showed that the tumor mass in the CAR-T cell-loaded lymph node group was significantly smaller than that in the other groups. Figure 8C The survival time of mice in different treatment groups was also monitored. The results showed that 50% of mice in the CAR-T-loaded lyophilized lymph node group survived for more than 90 days, significantly better than other control groups. Furthermore, the levels of IFN-γ and IL-2 in the tumors of the CAR-T-loaded lymph node group were also significantly increased. Figure 8E ).

[0078] Patient-derived model of pancreatic cancer recurrence:

[0079] Healthy female NSG immunodeficient mice (Southern Model Biotechnology Co., Ltd.) aged 6–8 weeks were used. Pancreatic cancer tissue obtained from surgically removed pancreatic cancer patients was implanted into NSG mice. When the tumor grew to 200 mm³, surgical resection was performed, leaving 20% ​​of the tumor as a residual tumor to simulate postoperative recurrence. Mice were treated postoperatively with PBS, lyophilized lymph nodes, CAR-T cell and cytokine-loaded gel, and CAR-T cell-loaded lyophilized lymph nodes, respectively. The CAR-T cell-loaded lyophilized lymph node group achieved effective control of tumor development, while other treatment groups only showed some allergic reaction. Figure 9A and Figure 9B ).

[0080] The nucleotide sequence encoding the CAR molecule (SEQ ID NO:1) is as follows:

[0081]

[0082] The amino acid sequence of the CAR molecule (SEQ ID NO:2) is as follows:

[0083] MLLLVTSLLLCELPHPAFLLIPDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRDYKDDDDKGSGEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPQVQLVESGGGVVQPGGSLRLSCATSGTIFSRNGMGWFRQAPGKQRELVARILNDGTTMYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCGYSDYRGTDYWGQGTQVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGL.

Claims

1. A delivery scaffold-cell complex, characterized in that, It includes a delivery scaffold and cells for treatment; The delivery stent was obtained by freeze-drying lymph nodes; Freezing involves placing the lymph nodes at -80°C or in liquid nitrogen. The cells used for treatment include engineered immune cells and stem cells.

2. The delivery scaffold-cell complex as claimed in claim 1, characterized in that, The lymph node is a human lymph node; and / or, the lymph node is a lymph node that does not contain metastatic tumor cells.

3. The delivery scaffold-cell complex as described in claim 1, characterized in that, The freeze-drying includes freezing and drying steps, wherein the freezing time is 2 min to 240 min; and / or, the drying time is 4 to 20 h.

4. The delivery scaffold-cell complex according to any one of claims 1-3, characterized in that, The engineered immune cells are selected from CAR-T cells, TCR-T cells, CAR-NK cells, and CAR-M cells.

5. The delivery scaffold-cell complex as described in claim 4, characterized in that, The engineered immune cells are CAR-T cells.

6. The delivery scaffold-cell complex as claimed in claim 1, characterized in that, The delivery scaffold-cell complex is prepared by injecting cells for treatment into the delivery scaffold.

7. The delivery scaffold-cell complex as claimed in claim 6, characterized in that, The concentration-to-mass ratio of the cells used for treatment to the delivery scaffold is (2 × 10⁻⁶). 6 (pcs / g); and / or, the cells used for treatment are centrifuged or precipitated and then resuspended in FBS-free X-Vivo medium or PBS, the suspension is drawn up using a syringe and injected into the delivery scaffold or the suspension is dripped directly into the scaffold.

8. The delivery scaffold-cell complex as claimed in claim 7, characterized in that, The resuspension and / or injection are performed on ice.

9. The delivery scaffold-cell complex as claimed in claim 1, characterized in that, When the cells used for treatment are CAR-T cells, the amino acid sequence of the CAR molecule is shown in SEQ ID NO:

2.

10. The delivery scaffold-cell complex as claimed in claim 9, characterized in that, The nucleotide sequence encoding the CAR molecule is shown in SEQ ID NO:

1.

11. Use of the delivery scaffold-cell complex as described in any one of claims 4-5 and 9-10 in the preparation of a medicament for inhibiting tumors.

12. The application as described in claim 11, characterized in that, The tumors are selected from stomach cancer, intestinal cancer, pancreatic cancer, cervical cancer, lung cancer, and prostate cancer.

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