Engineering probiotic-T cell chimera as well as preparation method and application thereof

Abstract

The invention discloses an engineering probiotic-T cell chimera as well as a preparation method and application thereof. Food-grade probiotic lactococcus lactis expressing Flt3L-OX40L double-cytokine fusion protein at the corresponding position of a thallus is successfully constructed in the early stage, then a dibenzocyclooctyne group (DBCO) can be expressed on the surface after chemical labeling, and meanwhile, an azide group (Azido) can be expressed on the surface after a T cell is labeled by glycometabolism. The two can be used for preparing the engineering probiotic-T cell chimera based on bio-orthogonal click chemistry. After successful construction, repeated experiments prove that the engineering probiotic-T cell chimera can promote T cell activation and differentiate into immune memory effector cells (TEM), and the mouse tumor tissue targeting capability of the engineering probiotic-T cell chimera is improved. Meanwhile, animal experiments indicate that the intravenous injection engineering probiotic-T cell chimera has a remarkable effect of inhibiting colorectal cancer tumors.

Description

Technical Field

[0001] The present invention relates to an engineered probiotic-T cell chimera and its preparation method and application, belonging to the fields of biomedicine and bioengineering. Background Art

[0002] In the past two decades, the development of immunotherapy has revolutionized the landscape of cancer treatment. The infusion of engineered T cells is a major component of immunotherapy, which can specifically recognize and attack tumor cells carrying specific antigens. Currently, chimeric antigen receptor (CAR)-T cells or T cell receptor (TCR)-T cells constructed by genetic engineering technology have shown good efficacy in clinical trials, but the serious adverse reactions caused by their "off-target effects" are inevitable.

[0003] To improve the tumor targeting of T cells, attention has been turned to the booming engineered bacteria technology. The food-grade probiotic Lactococcus lactis can utilize its facultative anaerobic characteristics to target tumor sites and colonize in tumors. To further reduce its inherent pathogenicity and enhance its immunogenicity, the recombinant plasmid pNZ8148-Flt3L-OX40L was used to make Lactis express the Flt3L-OX40L fusion protein, and an engineered Lactococcus lactis (FOLactis) expressing the Flt3L-OX40L bicytokine was successfully constructed, and a related patent (202111222043.0) has been obtained.

[0004] To achieve the improvement of the tumor targeting and function of FOLactis on T cells, a reliable, safe, synergistic and efficient chimeric drug delivery system can be constructed based on bioorthogonal click chemistry. Bioorthogonal click chemistry refers to rapid and selective chemical reactions under physiological conditions, which can occur in living cells or tissues without interfering with the biochemical processes of the organism itself. Among them, especially copper-free click chemistry (such as SPAAC and IEDDA), which does not require the use of toxic catalysts, is an ideal reaction system for bioconjugation. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide an engineered probiotic-T cell chimera and its preparation method and application.

[0006] Technical Solution: To solve the above technical problem, the present invention provides an engineered probiotic-T cell chimera, comprising DBCO-modified engineered probiotics and Azido-modified T cells; the DBCO-modified engineered probiotics and the Azido-modified T cells form a bridging structure with a 1,2,3-triazole ring as the core through bioorthogonal click chemistry; the DBCO-modified engineered probiotics are wrapped on the surface of the Azido-modified T cells through the bridging structure.

[0007] Among them, the engineered probiotic is Lactococcus lactis (FOLactis) that induces the expression of the pNZ8148-Flt3L-OX40L recombinant plasmid.

[0008] The present invention also provides a method for constructing the engineered probiotic-T cell chimera, comprising the following steps:

[0009] (1) Insert the gene shown in nucleotide sequence SEQ ID No.1 into an expression vector to prepare a recombinant plasmid; introduce the recombinant plasmid into competent cells, and after adding a DBCO-PEG2000-NHS solution, a covalent chemical reaction can be carried out to obtain DBCO-modified FOLactis;

[0010] (2) Add Ac4GalNAz to T cells, and perform sugar metabolism labeling to obtain Azido-modified T cells;

[0011] (3) Resuspend the DBCO-modified FOLactis in step (1) and the Azido-modified T cells in step (2) in a medium without antibiotics and react to obtain the engineered probiotic-T cell chimera.

[0012] Among them, the recombinant plasmid in step (1) can express a protein composed of the amino acid sequence shown in SEQ ID No.2. Among them, SEQ ID No.2: HHHHHHPVGLIGGTPDCYFSHSPISSNFKVKFRELTD HLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPRPRQPVGLIGSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL*

[0013] Among them, the T cells in step (2) are amplified murine T cells.

[0014] The present invention chemically labels FOLactis to express dibenzocyclooctyne group (DBCO) on its surface. Meanwhile, after the T cells are labeled by sugar metabolism, azide group (Azido) can be expressed on their surface. Based on bioorthogonal click chemistry, an engineered probiotic-T cell chimera can be prepared from the two. The present invention enhances the activity and targeting function of the infused T cells, and at the same time avoids the systemic toxicity caused by the large-dose application of bacteria, and can realize the combined mode of T cell infusion and bacterial co-delivery.

[0015] The present invention also provides the application of the engineered probiotic-T cell chimera in the preparation of a drug for treating cancer.

[0016] Among them, the cancer includes colon cancer, gastric cancer, breast cancer, liver cancer, lung cancer or cervical cancer.

[0017] Among them, the engineered probiotic-T cell chimera activates T cells by promoting.

[0018] Among them, the engineered probiotic-T cell chimera promotes the infiltration of the infused T cells into tumor cells.

[0019] The present invention also provides a drug for treating cancer, which contains the engineered probiotic-T cell chimera.

[0020] Among them, the cancer includes colon cancer, gastric cancer, breast cancer, liver cancer, lung cancer or cervical cancer.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention connects FOLactis with targeting and immune-stimulating functions and T cells playing an anti-tumor immune role through bioorthogonal click chemical reaction, which has the characteristics of rapidity and high efficiency; it can promote the effective activation of T cells, and at the same time promote the differentiation of T cells themselves into immune memory effector cells (TEM), and its ability to target mouse tumor tissues is improved, providing a new method for the improvement and optimization of T cell infusion and the treatment of tumor-related diseases. Description of the Drawings

[0022] Figure 1 Confocal imaging of FOLactis modified with DBCO;

[0023] Figure 2 Confocal imaging of T cells modified with Azido;

[0024] Figure 3 Flow cytometry data of T cells modified with Azido;

[0025] Figure 4 Confocal imaging of the engineered probiotic-T cell chimera;

[0026] Figure 5Flow cytometry data for quantification of engineered probiotic-T cell chimeras;

[0027] Figure 6 Scanning electron microscopy images of engineered probiotic-T cell chimeras;

[0028] Figure 7 Flow cytometry results for the proliferation performance of engineered probiotic-T cell chimeras after construction;

[0029] Figure 8 For engineered probiotic-T cell chimeras promoting T cell (CD4 + CD69 + 、CD4 + CD25 + 、CD8 + IFN-γ + ) activation flow cytometry data;

[0030] Figure 9 Flow cytometry data for engineered probiotic-T cell chimeras promoting the differentiation of T cells into TEM cells (CD3+CD8+CD62L-CD44+);

[0031] Figure 10 In vitro near-infrared imaging of the ability of engineered probiotic-T cell chimeras to enhance targeting of mouse tumor tissues (from left to right are lymph nodes, spleen, liver, heart, tumor, kidney, lung);

[0032] Figure 11 Average tumor growth curves of mice after different treatments;

[0033] Figure 12 Tumor growth curves of each mouse in different treatment groups. Detailed implementation manners

[0034] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0035] 1. Construction of DBCO-modified FOLactis

[0036]

[0037] Meanwhile, Lactococcus lactis (Lactis) was inoculated into GSGM-17 liquid medium for culture, and bacteria were collected during the exponential growth phase to obtain Lactis competent cells. Lactis competent cells were mixed with the recombinant plasmid, and the recombinant plasmid was introduced into Lactis competent cells by electroporation. Then the bacterial solution was inoculated into the recovery medium (M17 medium + 0.5% glucose + 10 μg / mL chloramphenicol) and incubated for 2 h, and then the bacterial solution was inoculated into GM17 solid medium for culture for 48 h, and the colonies were FOLactis.

[0038] (2) DBCO modification of FOLactis: Take 1×10 9 CFU FOLactis, make up the volume to 1 mL with PBS, add an appropriate amount of NaOH standard solution, adjust the pH to 8, and then add DBCO-PEG2000-NHS dissolved in DMSO to make its final concentration 100 μM. React with shaking at room temperature for 2 h. Centrifuge at 8000 rpm for 7 min, discard the supernatant, and wash the precipitate with PBS 3 times to obtain DBCO-modified FOLactis.

[0039] 2. Construction of azido-modified T cells, specifically including:

[0040] (1) Acquisition and activation of murine T cells: Dilute anti-CD3 antibody (Thermo Fisher, 16-0032-82) with sterile PBS to a final concentration of 1 μg / mL, coat the anti-CD3 antibody in a six-well plate, and incubate the six-well plate at 37 °C for 2 h. After 2 h, aspirate and discard the anti-CD3 solution. After C57BL / 6 mice were sacrificed by cervical dislocation, they were soaked in 75% alcohol for 15 min, the spleen was surgically removed with sterile scissors and forceps, placed in a six-well plate containing normal saline, and ground in a laminar flow hood until the tissue turned white. Filter the cell suspension through a 40 μm filter, centrifuge at 350×g for 5 min, and discard the supernatant. Resuspend the cells with 1 - 2 mL of red blood cell lysate (1x RBC lysate), let stand for 5 min, centrifuge at 350×g for 3 min, and discard the supernatant. Rinse once with 10 mL of PBS, centrifuge at 350×g for 5 min, discard the supernatant, resuspend with 5 - 6 mL of RPMI1640 culture medium containing 20 ng / ml IL-2 (Thermo Fisher, PHC0026) and 3 μg / mL anti-CD28 (Thermo Fisher, 16-0281-82), and inoculate into the treated six-well plate for culture at 37 °C, and change the medium by half the next day.

[0041] (2) Azido modification of T cells: After culturing T cells for 3 - 4 days, Ac4GalNAz was added to a final concentration of 50 μM, and the cells were cultured for another 2 days. Then, the cells were centrifuged at 350×g for 5 min, and the supernatant was discarded to obtain Azido - modified T cells.

[0042] 3. Synthesis of engineered probiotic - T cell chimera: The DBCO - modified FOLactis obtained in step 1 and the Azido - modified T cells obtained in step 2 were resuspended in 1 mL of RPMI 1640 culture medium without antibiotics at a ratio of 50:1, and reacted at 37 °C for 2 hours. Then, the cells were centrifuged at 350×g for 5 min, and the supernatant was discarded to obtain the engineered probiotic - T cell chimera (T@FO).

[0043] Result analysis:

[0044] I. Product characterization

[0045] Take the DBCO - modified FOLactis, add the N3 - TAMRA solution (dissolved in DMSO) to a final concentration of 10 μM of N3 - TAMRA in the total system. After incubating in the dark at room temperature for 1 h, wash 3 times with PBS, and then take pictures using a laser confocal scanning microscope. As Figure 1 shown, compared with the control group treated with PBS, FOLactis treated with 50 μM or 100 μM DBCO - PEG2000 - NHS solution showed stronger N3 - TAMRA fluorescence, indicating that the DBCO group was successfully modified on the surface of FOLactis.

[0046] Take the Azido - modified T cells, add the DBCO - Fluor 488 solution dissolved in DMSO to a final concentration of 10 μM of DBCO - Fluor488 in the total system. After incubating in the dark at room temperature for 0.5 h, wash 3 times with PBS, fix with 4% paraformaldehyde, add DAPI staining solution at 3 times the volume of the sample to be tested, wash 3 times with PBS, and then take pictures using a laser confocal scanning microscope and perform flow cytometry detection. As Figure 2 shown, compared with the control group treated with PBS, T cells treated with 50 μM or 100 μM Ac4GalNAz showed stronger DBCO - Fluor 488 fluorescence. At the same time, as Figure 3 shown, the fluorescence intensity on the surface of T cells tended to increase with the increase of the modification concentration, suggesting that the Azido group was successfully modified on the surface of T cells.

[0047] Take DBCO-modified FOLactis, add the CY5 solution dissolved in DMSO, so that the final concentration of CY5 in the total system is 10 μM, incubate in the dark at room temperature for 1 h, and wash 3 times with PBS. Take azido-modified T cells, add the CFSE solution, so that the final concentration of CFSE in the total system is 1 μM, incubate at 37 °C for 10 minutes, add 10 times the volume of physiological saline to terminate the staining respectively, and wash 3 times with PBS. Resuspend the separately stained FOLactis and T cells in 1 mL of 1640 medium without antibiotics, react at 37 °C for 2 hours, centrifuge at 350 × g for 5 min, discard the supernatant, and take pictures with a laser confocal scanning microscope, perform flow cytometry detection and scanning electron microscope imaging. As Figure 4 and Figure 6 shown, FOLactis is conjugated to the surface of T cells through bioorthogonal click chemical reactions. As Figure 5 shown, only when both FOLactis and T cells are labeled, 5×10 7 FOLactis can cover 10 6 activated cells at a level of 59.7%. Among them, T+FO in the figure represents the mixture of unmodified T cells and unmodified FOLactis, T+DBCO-FO represents the mixture of unmodified T cells and DBCO-modified FOLactis, N3-T+FO represents the mixture of azido-modified T cells and unmodified FOLactis, and N3-T+DBCO-FO represents the mixture of azido-modified T cells and DBCO-modified FOLactis.

[0048] II. Cytotoxicity study of T@FO

[0049] Seed mouse T cells into 96-well plates (2×10 5 cells / well), after culturing for 48 h, add the CFSE solution, so that the final concentration of CFSE in the total system is 1 μM, incubate at 37 °C for 10 minutes, add 10 times the volume of physiological saline to terminate the staining, wash 3 times with PBS, discard the supernatant, dilute the drug with pure 1640 medium, and divide it into 3 groups, namely (1) T cell group; (2) T cell + beads (activated magnetic beads) group; (3) T@FO + beads group. Perform flow cytometry detection on the 4th day after culturing, and the results show that the construction of engineered probiotic-T cell chimeras does not impair cell viability, phenotype or effector function ( Figure 7 ).

[0050] III. Immune activation effect of T@FO

[0051] Seed mouse T cells into 96-well plates (2×10 5(cells / well), after culturing for 48 h, discard the supernatant, dilute the drug with pure 1640 medium, and divide it into 3 groups, namely (1) normal saline (NS) group; (2) T cell + FOLactis (T+FO) group; (3) T@FO group. The concentration is quantified by the bacteria in T@FO, and added to each well at a ratio of 1:50 of T cells to FOLactis. After co-incubating with T cells for 24 h, the activation level of T cells is detected by flow cytometry.

[0052] As Figure 8 shown, compared with other groups, the activation effect of the T@FO group on T cells was significantly increased, demonstrating that the engineered probiotic-T cell chimera prepared by bioorthogonal click chemical reaction can successfully promote the activation of T cells through bacterial therapy. As Figure 9 shown, T cells differentiated into immune memory effector cells (T EM ), and the TEM in the T@FO group was significantly increased.

[0053] IV. Targeting effect of T@FO

[0054] Mix DIR dye with T cells or T@FO at a volume ratio of 1:100, let it stand at 30 °C for 30 min, centrifuge at 350×g for 5 min, and repeat washing 3 times to retain the precipitate (DIR-T cells, DIR-T@FO).

[0055] Select 4-5-week-old BALB / c mice, and subcutaneously inject 4T1 cells (3×10 5 cells / mouse). After the tumor grows to about 80 mm 3 , randomly divide the mice into 2 groups, namely (1) DIR-T cells (1×10 7 / mouse); (2) DIR-T@FO (1×10 7 / mouse) for administration. The administration protocol is to inject once via the tail vein after tumor formation, and dissect the mice 6 h after administration to collect tumor tissues for near-infrared imaging. As Figure 10 shown, T@FO promotes the infiltration of infused T cells into tumor cells.

[0056] V. In vivo verification of the tumor suppression effect of intravenous injection of T@FO

[0057] Select 4-5-week-old BALB / c mice, and subcutaneously inject CT26 cells (1×10 6 cells / mouse). When the tumor grows to about 100 mm 3 (length mm × width mm × width mm / 2), administer drugs. Randomly divide the mice into 4 groups, namely ① normal saline (NS) group; ② T cells (1×10 7 T cells / mouse); ③ T cells + FOLactis (1×10 7 T cells + 2×108 FOLactis / per mouse); ④ T@FOLactis (1×10 7 T@FOLactis per mouse). In a dosing regimen of once every 3 days for a total of 3 times, the mouse colorectal cancer model was injected via the tail vein. The tumor diameter was measured every other day, and the tumor growth curve and mouse survival curve were plotted to clarify the anti-tumor activity of T@FOLactis.

[0058] As Figure 11 and 12 shown, in the subcutaneous colorectal cancer mouse model, the engineered probiotic-T cell chimera can significantly inhibit tumor growth, and the complete remission rate (CR) can reach 20%.

Claims

1. An engineered probiotic-T cell chimera, characterized in that, Comprising DBCO-modified engineered probiotics and azido-modified T cells; the DBCO-modified engineered probiotics are wrapped on the surface of azido-modified T cells through a bridging structure with a 1,2,3-triazole ring as the core.

2. The engineered probiotic-T cell chimera according to claim 1, characterized in that, The engineered probiotics comprise Lactococcus lactis.

3. A method for constructing the engineered probiotic-T cell chimera according to claim 1, characterized in that, Comprising the following steps: (1) Insert the gene with the nucleotide sequence shown in SEQ ID No.1 into an expression vector to prepare a recombinant plasmid; introduce the recombinant plasmid into competent cells, and after adding a DBCO-PEG2000-NHS solution, carry out a covalent chemical reaction to obtain DBCO-modified FOLactis; (2) Add Ac4GalNAz to T cells and carry out sugar metabolism labeling to obtain azido-modified T cells; (3) Resuspend the DBCO-modified FOLactis in step (1) and the azido-modified T cells in step (2) in a medium without antibiotics and react to obtain an engineered probiotic-T cell chimera.

4. The construction method according to claim 3, characterized in that, The T cells in step (2) are amplified murine T cells.

5. Use of the engineered probiotic-T cell chimera according to claim 1 or 2 in the preparation of a medicament for treating cancer.

6. The application according to claim 5, wherein The cancer includes colon cancer, gastric cancer, breast cancer, liver cancer, lung cancer or cervical cancer.

7. The application according to claim 5, characterized in that, The engineered probiotic-T cell chimera treats cancer by promoting T cell activation.

8. The application according to claim 5, characterized in that The engineered probiotic-T cell chimera promotes the infiltration of the infused T cells into tumor cells.

9. A drug for treating cancer, characterized in that, It contains the engineered probiotic-T cell chimera according to claim 1.

10. The drug according to claim 9, characterized in that, The cancer includes colon cancer, gastric cancer, breast cancer, liver cancer, lung cancer or cervical cancer.

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