Ligand-targeted cell conjugate (LTCC)-based anti-tumor immune cell

Abstract

Non-natural sugars modified with a bioorthogonal reactive group are added into a culture medium of immune cells such as NK cells to obtain immune cells modified with the bioorthogonal reactive group; and then, under a physiological condition, a targeting ligand, for example, a nanobody, is modified to a surface of each of the immune cells through a bioorthogonal reaction, wherein the targeting ligand has one terminal with a bioorthogonal reactive pairing group which is capable of being matched and connected with the bioorthogonal reactive group to generate a connecting reaction, connection is implemented by a transpeptidase SrtA-mediated chemoenzymatic method. The targeting ligand highly specifically recognizes and binds to a highly expressed receptor on the surface of tumor cells. The immune cell modified with the targeting ligand can specifically bind in a targeted way to the tumor cells, therefore generating cytokines, killing and damaging tumor cells.

Claims

1. A ligand-targeted cell conjugate (LTCC)-based anti-tumor immune cell, characterized in that an immune cell modified with a bioorthogonal reactive group on a surface and a targeting ligand modified with a bioorthogonal reactive pairing group on the surface are correspondingly connected through a bioorthogonal reaction, to form a targeting immune cell, which is conjugated with the targeting ligand on the surface, wherein the targeting ligand performs specific recognition and is connected with a highly expressed receptor on the surface of the tumor cell; wherein the targeting ligand is Nanobody 7D12 (Nb 7D12); the targeting immune cell is Natural Killer cell (NK cell); the bioorthogonal reactive group is an azide group; and the bioorthogonal reactive pairing group is dibenzocyclooctyne (DBCO).

2. The LTCC-based anti-tumor immune cell according to claim 1, wherein the NK cells comprise an NK92-MI cell line, an NK 92 cell line or primary NK cells isolated from human bodies.

3. The LTCC-based anti-tumor immune cell according to claim 1, wherein the bioorthogonal reactive pairing group and the bioorthogonal reactive group on the non-natural sugars are used in pairs.

4. The LTCC-based anti-tumor immune cell according to claim 3, wherein the non-natural sugar is N-azidoacetylmannosamine-tetraacylated (Ac.sub.4ManNAz).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the principle process according to the invention.

(2) FIG. 2 is a schematic diagram of screening of an optimal Ac.sub.4ManNAz concentration; A is a displacement diagram of flow cytometry, and B is a fluorescence quantitative analysis diagram.

(3) FIG. 3 is a schematic diagram of influences of Ac.sub.4ManNAz on activities of NK92-MI cells.

(4) FIG. 4 is schematic diagram of a functionalized targeting ligand with a Nb synthesized by a chemoenzymatic method; A is a full flowchart of chemical synthesis of DBCO-PEG4-GGG, B is a flowchart of synthesis of DBCO-PEG4-7D12 by connection of the DBCO-PEG4-GGG and a Nb 7D12 using sortaseA, C is a schematic diagram of mass spectrum identification of a connected product DBCO-PEG4-7D12 (theoretical molecular weight is 16391.5; and an observed value of mass spectrum is 16390.4).

(5) FIG. 5 is schematic diagram of detection after cells are loaded with the Nb 7D12; A is a schematic diagram of observation by laser confocal microscopy, and B is a schematic diagram of screening of the optimal concentration for loading of the DBCO-PEG4-7D12.

(6) FIG. 6 is a schematic diagram of in-vitro targeting property validation of 7D12-NK92MI cells; A is a schematic diagram of affinity between cancer cells with positive EGFR and NK92-MI cells. B is a schematic diagram of connection between cancer cells with positive EGFR and 7D12-NK92MI cells, and C is a statistical diagram of the connection rate of the above-mentioned groups.

(7) FIG. 7 is a schematic diagram of in-vitro anti-tumor activities of NK92-MI cells modified with the Nb; A is a schematic diagram of comparison of anti-tumor activities at different effect-target ratios, and B is a schematic diagram of essentiality validation of the Nb in cell-killing activities.

(8) FIG. 8 is a schematic diagram of broad anti-tumor spectrum analysis of NK92-MI cells modified with the Nb.

(9) FIG. 9 is a schematic diagram of in-vivo anti-tumor activities of NK92-MI cells modified with the Nb; A is a flowchart of an animal experiment, B is a schematic diagram of real tumors before and after treatment, C is a schematic diagram of changes in the tumor volume during treatment, D is a schematic diagram of comparison of tumor weights before and after the treatment, E is a schematic diagram of a tumor inhibition rate before and after the treatment, and F is a schematic diagram of changes in mouse weight during treatment.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

(10) To facilitate understanding of the technical means, objectives and effects of the invention, the invention is further described with reference to, but not limited by, the following embodiments.

Embodiment

(11) The invention can be better understood according to the following embodiments. However, it is understandable for a skilled in the art that, the specific material proportions, technological conditions and results described in the embodiments apply to the invention only, and should not be construed as limiting the protective scope of the invention.

(12) An objective of the invention is to disclose a preparation method and use of an LTCC-based anti-tumor immune cell. In the embodiments, a Nb 7D12, as a targeting ligand, and NK cells, as examples of immune cells, are used to fully interpret and describe preparation and application of the anti-tumor NK cells using NBCC. NK cells used belong to an NK92-MI or NK92 cell line which is generally called off-the-shelf reagent. Such approach facilitates cell acquisition, cultivation and augmentation in a large scale, and subsequent conversion into production. The following embodiments are not limited to the two types of NK cells, and immune cells including other NK cells, T cells and macrophages can also be used; similarly, the targeting ligand is also not limited to the Nb 7D12, and can be other Nbs or macromolecules with targeting and specific recognition functions, for example, aptamers, scFv, glycoside ligands, target peptides, etc.

Embodiment 1: Preparation of a Functionalized Nb

(13) Preparation of a functionalized Nb includes three procedures, namely fermentation and purification of a Nb, chemical synthesis of a bioorthogonal reactive group which carries a triglycine peptide, and Sortase A enzyme-mediated connection.

(14) Fermentation and Purification of the Nb:

(15) 100 L of previously prepared glycerol frozen bacteria of engineered Escherichia coli which were able to produce a Nb 7D12 (targeting at EGFR) was inoculated in 25 mL of an LB culture medium which contained 100 g/mL kanamycin, and the bacteria in the LB culture medium were cultured and activated at 37 C. and at 200 rpm overnight. On the second day, 2% of the bacteria were transferred into a TB culture medium which contained 100 g/mL kanamycin and cultured at 37 C. and at 200 rpm, wherein the formula of the TB culture medium included 12 g/L peptone, 24 g/L yeast powder, 5 g/L glycerol, 16.4 g/L K.sub.2HPO.sub.4.3H.sub.2O and 2.31 g/L KH.sub.2PO.sub.4. After OD.sub.600 reached 0.6 to 0.8, IPTG with a final concentration of 0.2 mM was added to induce expression at 16 C. for 24 hours. After fermentation was ended, the bacteria were collected through centrifugation at 8000 rpm. An ultrasonication lysis buffer was added for ultrasonication (the buffer was prepared according to the description of a nickel column required subsequently); then centrifugation was carried out at 12000 rpm and 4 C. for 15 min; and supernatant was collected and repeatedly centrifuged for 3 times. Subsequently, the nickel column was placed in the supernatant to purify the Nb 7D12, and finally, a gel column G25 for desalination was used to remove imidazolium salt.

(16) Chemical Synthesis:

(17) 1) Synthesis of azido sugars: 1.22 g of mannosamine hydrochloride was dissolved in 50 mL of MeOH; then, 1.06 mL of 30% NaOMe/MeOH solution was added; next, the mixed materials were stirred for 1 hour at room temperature; later, 0.815 mL of TEA and 2.9 g of chloroacetic anhydride were added; next, the mixed substance was stirred and reacted at room temperature for 6 hours; the obtained solution was eluted by gradient using CH.sub.2Cl.sub.2 and MeOH at a ratio from 6:1 to 5:1 through the gel column to obtain N-chloracetyl mannosamine; after a solvent volatilized, 16 mL of DMF and 3.68 g of NaN.sub.3 were added; the solution reacted at 80 C. for 4 hours; after the solvent volatilized as the solution cooled to room temperature, 30 mL of pyridine and 15 mL of acetic anhydride were added; the solution was stirred at room temperature and was kept overnight for reaction; then, 200 mL of ethyl acetate was added into the reacting solution; the mixed solution was mixed well; 100 mL of 1M hydrochloric solution was added into an organic layer for washing; the solution was separated after three times of washing; next, 100 mL of saturated NaHCO.sub.3 was added to wash the solution for three times, then the solution was separated again; 100 mL of saturated NaCl was added to wash the solution twice, and then the solution was separated one more time; next, anhydrous Na.sub.2SO.sub.4 was added to dry the substance, and the dried substance was eluted by gradient using n-hexane and ethyl acetate at a ratio from 1:1 to 1:2 through the gel column to obtain the product Ac.sub.4ManNAz.

(18) In addition to the previously prepared N-azidoacetylmannosamine-tetraacylated (Ac.sub.4ManNAz), non-natural sugars applicable to the invention further include: N-azidoacetylgalactosamine-tetraacylated (Ac.sub.4GalNAz), N-azidoacetylglucosamine-tetraacylated (Ac.sub.4GlcNAz), N-azidoacetylmannosamine-acetylated (ManNAz), N-azidoacetylneuraminic acid (SiaNAz), N-levulinoylmannosamine (ManLev), and N-propionylmannosamine-acetylated (Ac.sub.4ManLev), etc., wherein parent materials of corresponding sugars are respectively used as raw materials. Similarly, those sugars can also be prepared through above-mentioned chemical synthesis.

(19) According to the embodiments of the invention, as shown in FIG. 2, it can be seen that the optimal concentration of the added non-natural sugars is 50 to 100 M with reference to the displacement effect of the flow cytometry and results of the quantitative fluorescence analysis.

(20) 2) Synthesis of DBCO-PEG4-GGG: the entire process can be seen in FIG. 4A. Ethylenediamine with one terminal protected by a Cbz group and triglycine peptide with one terminal protected by a Boc group jointly performed an amide condensation reaction at an equivalent proportion of 4:5 to 4:7; after removal of the Cbz group, the reacting product reacted with DBCO-PEG4-NHS at an equivalent proportion of 2:1 to 1:1; and finally, the product DBCO-PEG.sub.4-GGG was obtained after removal of the Boc group.

(21) The specific method is as follows:

(22) 310 mg of Cbz-EDA-NH.sub.2, 578 mg of Boc-GGG-COOH (Boc: t-Butyloxy carbonyl), 411 mg of HOBt, 0.6 mL of TEA, and 573 mg of EDC.HCl were mixed well in 10 mL of DMF at 0 C.; the mixed materials were stirred at room temperature for 4 hours; then, DCM and saturated NaCl were used for extraction and liquid separation; then, anhydrous Na.sub.2SO.sub.4 was added for drying; next, the dried substance was purified through silica column chromatography using a chromatographic liquid which was composed of DCM and MeOH at a ratio of 50:1; then, 20 mL of MeOH and 120 mg of Pd/C were added; the mixed substance was stirred with the existence of hydrogen gas at room temperature for 12 hours for removal of the Cbz group; and Boc-GGG-EDA-NH.sub.2 was obtained after purification and spin drying. 70 mg of Boc-GGG-EDA-NH.sub.2 was dissolved in 10 mL of DMF; then, 140 mg of DBCO-PEG.sub.4-NHS was added; the mixed material was stirred for reaction for 30 min at room temperature; after the reaction ended, the reacting product was purified through silica column chromatography using a chromatographic liquid which was composed of DCM and MeOH at a ratio of 10:1. Then, 20% TFA/DCM was added, and the Boc group was removed by stirring stirred for 1.5 hours at room temperature; finally, a proper amount of toluene was added, and reduced pressure distillation was carried out to obtain DBCO-PEG.sub.4-GGG-NH.sub.2.

(23) Sortase A enzyme-mediated conjugation: the entire process can be seen in FIG. 4B. The Nb was expressed by Escherichia coli, and a carboxyl terminal carried a site capable of being recognized by LPXTG (X was any amino acid, for example E/A/V, etc.) transpeptidase Sortase A, and a histidine tag (His tag). During Sortase A enzyme-mediated connection, 20-25 M of the targeting ligand, i.e. the Nb, 250-500 M of micromolecular substrate containing a bioorthogonal reactive group, and 3-5 M of transpeptidase Sortase A were mixed and reacted at 16 C. for 5-9 hours in a Tris-HCl/NaCl buffer which had a pH value of 7.4-7.5 and contained CaCl.sub.2). Then, the reaction product was purified using magnetic beads coated with nickel-ion or agarose column bearing nickel-ion; and finally, the gel column G25 was used to remove micromolecules such as the bioorthogonal reactive group by steps of dechlorination.

(24) The reaction system included: 20 M of 7D12, 500 M of DBCO-PEG.sub.4-GGG-NH.sub.2, 5 M of enzyme Srt A, and 1Srt A reaction buffer solution (formula: 50 mM Tris, 150 mM NaCl, and 5 mM CaCl.sub.2), pH in the range of 7.4-7.5). Materials were added according to the above system. After being mixed well, the materials reacted at 16 C. for 5 hours; then, 7D12, Srt A and cut His tag, which did not react, were extracted using magnetic beads coated with nickel-ion; the supernatant was collected and eluted with the gel column G25 to remove excessive DBCO-PEG.sub.4-GGG-NH.sub.2, and then DBCO-PEG.sub.4-7D12 was obtained.

Embodiment 2: Influences of Azido Sugars on Activities of NK Cells

(25) This embodiment describes validation of influences of the added non-natural sugars on cell activities. The specific method is as follows: non-natural sugars were absorbed by cells; the cells were cultured for a period of time; then, OD.sub.450 was determined using a cell counting kit 8 (CCK8) (Beyotime), and a difference in cell activities of a group added with sugars and a group without sugars was analyzed.

(26) The specific process is as follows: 110.sup.5 pieces of NK cells were laid in a 96-pore plates; 0.1% DMSO was added into a control group, while Ac.sub.4ManNAz with a final concentration of 50 M (the mount of the DMSO was equivalent to 0.1%) was added into an experimental group; then, the cells were cultured in an incubator at 37 C., and subsequently added with the CCK8 after 24 hours and 48 hours respectively; and after 2-hour incubation, OD.sub.450 was detected using a microplate reader. The results, as shown in FIG. 3, show that, the added non-sugars were not toxic to cells, but slightly promoted growth.

Embodiment 3: Modification of Immune Cells Such as NK Cells with Nb

(27) This embodiment describes acquisition of NK cells with an azide tag and modification with 7D12. The principle process is as shown in FIG. 1. Non-natural sugars were absorbed; a bioorthogonal reactive group which modified the sugars were loaded to cell surfaces through glucose metabolism engineering processes including glucolysis and sialic acid metabolism; then cells were collected, and under a condition similar to the physiological condition, added with the functionalized Nb (110.sup.4 pieces of NK cells treated using 8-10 g of functionalized Nb), and the mixture reacted at 37 C. for 1-1.5 hours. Whether the cells were successfully modified with the Nb was validated through immunofluorescence.

(28) The specific method is as follows:

(29) The previously chemically synthesized Ac.sub.4ManNAz was dissolved in DMSO to prepare a 50 mM parent solution; after filtration and sterilization, 1 of the mixed solution was added into a special culture medium special for NK cells to enable the culture medium to contain 50 M non-natural sugars; NK92-MI cells were cultured in the culture medium for 48 hours and then centrifuged at 600 rpm for 5 min; and, the cells were collected and washed with PBS for 3 times to obtain NK cells modified with the bioorthogonal reactive group N3 (N3-NK92MI).

(30) Next, 8.2 g of DBCO-PEG.sub.4-7D12 was added into every 10000 pieces of cells; the mixed substance reacted at 37 C. for 1.5 hours in the PBS system; and finally, the reacting product was washed with the PBS for 3 times to obtain the functionalized NK cells with 7D12 (7D12-NK92MI).

(31) The obtained functionalized NK cells (7D12-NK92MI) were displayed through immunofluorescence, as shown in FIG. 5, which represented that modification with 7D12 succeeded.

Embodiment 4: In-Vitro Targeting Detection of the Nb-Functionalized Immune Cells.Nb

(32) This embodiment describes analyses of the in-vitro targeting property and anti-tumor activities of the Nb-functionalized immune cells Nb(7D12-NK92MI) obtained in Embodiment 3. The specific method is as follows: NK cells and targeted tumor cells were marked with two colors of fat-soluble dyes respectively; after adherence of the targeted tumor cells, NK cells which were not modified with the Nb and NK cells which were modified with the Nb were respectively cultured together with the targeted tumor cells at an effect-target ratio of 1:1, and the tumor cell adhering ability of the immune cells modified with the Nb was observed through fluorescence imaging.

(33) The following process is as follows:

(34) First, NK92MI/7D12-NK92MI cells were marked with green fluorescent DiO, and high-EGFR-expression LOVO cells which are kept overnight for adherence were marked with red fluorescent DiR; then, NK cells and LOVO cells were mixed at a ratio of 1:1 and cultured for 2 hours; the obtained product was washed with the PBS, fixed, and then re-washed with the PBS, and finally imaged through laser confocal.

(35) Results show that, the rate of connection between the 7D12-NK92MI cells and the LOVO cells is obviously higher than that between the NK92MI cells and the LOVO cells (FIG. 6), reaching about 48%. The results indicate that, the 7D12 is able to greatly enhance the tumor-binding ability of the NK92MI cells, and the functionalized immune cells modified with the Nb have higher tumor cell adhering ability.

Embodiment 5: In-Vitro Anti-Tumor Activity Detection of the Functionalized Immune Cells with the Nb

(36) This embodiment describes analyses of the in-vitro anti-tumor activity detection of the functionalized immune cells (7D12-NK92MI) with the Nb obtained in Embodiment 3. The specific method is as follows: NK cells which were not modified with the Nb and NK cells which were modified with the Nb were respectively cultured together with the targeted tumor cells at different effect-target ratios; the supernatant was collected and cultured; and the tumor cell killing ability of the immune cells was determined through LDH (lactic dehydrogenase).

(37) The specific process is as follows: NK92MI cells or 7D12-NK92MI cells were cultured together with LOVO cells at the effect-target ratios of 1:5, 1:1 and 5:1 respectively; and then, and the cell-killing ability of NK cells was determined through CytoTox 96 Non-Radioactive Cytotoxicity Assay. As shown in FIG. 7, as the effect-target ratio rises, the tumor-killing ability obviously increases, and the activity of the NK92MI cells modified with the 7D12 is apparently higher than that of the NK92MI cells without the 7D12.

(38) Then, LOVO cells which highly expressed EGFR and SW620 cells which lowly expressed EGFR were used to analyze whether the increase in the tumor-killing ability resulted from the Nb 7D12. As shown in FIG. 7B, NK92MI cells which are modified with the 7D12 and NK92MI which are not modified with the 7D12 are not obviously different in SW620 killing ability, while the LOVO cell-killing activity of the 7D12-NK92MI cells is obviously higher than that of the NK92MI cells.

(39) In addition, through competitive analysis (free 7D12) as shown in FIG. 7B, LOVO cells were treated with free Nb 7D12, and then the treated cells were cultured together with 7D12-NK92MI cells. The result indicates an obvious decline in tumor-killing activity.

(40) Several other types of high-EGFR-expression tumor cells, for example, MDA-MB-468 (mammary cancer), A431 (cutaneous squamous carcinoma), A549 (lung cancer) were used to analyze the anti-tumor spectrum of the 7D12-NK92MI cells. The results as shown in FIG. 8 indicate that NK92MI cells engineered with the 7D12 are able to obviously skill the several types of cancer cells.

(41) Results of the embodiment show that, NK cells modified with the Nb are superior to NK cells which are not modified the Nb in the ability of killing various tumor cells, and have a broad anti-tumor spectrum at the same time.

Embodiment 6: In-Vivo Anti-Tumor Activity of the Functionalized Immune Cells with the Nb

(42) In this embodiment describes detection of the in-vivo anti-tumor activity of the functionalized immune cells with the Nb (7D12-NK92MI) obtained in Embodiment 3. The specific method is as follows.

(43) NOD-SCID mice without NK cells were used as models and were subcutaneously injected with 0.1 mL LoVo cell suspension (3 million cells). Treatment began after the tumor volume reached 15 mm.sup.3. A PBS was used a blank control group and NK92MI cells were used as a negative control group. Each of the mice was injected with 3 million pieces of treating cells via the caudal vein. NK cells which were not modified with the Nb and NK cells which were modified with the Nb were respectively injected via the caudal vein by a number equal to or more than the number of tumor cells during tumor transplantation for a total of 5 times. The injection was given once every two days.

(44) The tumor volume and mouse weight were determined regularly (as shown in FIGS. 9C, F). After two consecutive weeks of treatment, the average tumor volumes of the experimental group, the control group and the PBS group were 410.5 mm.sup.3, 647.5 mm.sup.3 and 665.7 mm.sup.3, respectively. In the entire treatment process, the weight of each of the mice was not obviously changed. Then, the mice ended with euthanasia. Tumors were taken from the mice and weighed (as shown in FIGS. 9B, D). Calculation results showed that the average tumor weights of the experimental group, the control group and the PBS group were 232.8 mg, 338.7 mg and 329.1 mg, respectively. In addition, results showed that, the tumor growth inhibition rate of the NK92MI cells which were not modified with the Nb 7D12 was 7.4%, while the tumor growth inhibition rate of the NK92MI cells which were modified with the Nb 7D12 reached 44%, indicating that the treatment effects of the NK92MI cells engineered with the Nb 7D12 were better (FIG. 9E). Therefore, the treatment group of NK cells which were modified with the Nb had a higher tumor inhibition rate and did not impose great influences on the weight of the mice, indicating that NK cells modified with the Nb achieved a better treatment effect and had higher biological safety at the same time.

(45) The invention discloses an LTCC-based anti-tumor immune cell, a preparation method and use thereof, and belongs to the field of biomedical engineering. Non-natural sugars modified a bioorthogonal reactive group (for example, an azide group, a ketone group) are added into a culture medium of immune cells such as NK cells to obtain immune cells modified with the bioorthogonal reactive group; and then, under a physiological condition, a surface of each of the immune cells is modified with the targeting ligand, for example, a Nb, through the bioorthogonal reaction, wherein the targeting ligand has one terminal with a bioorthogonal reactive pairing group (dibenzocyclooctyne (DBCO), hydroxylamine), which is capable of matching and connecting with the bioorthogonal reactive group, the targeting ligand and the bioorthogonal reactive pairing group are connected by a transpeptidase SrtA-mediated chemoenzymatic method disclosed in the invention, and the targeting ligand has features of high specific recognition and connection with a highly expressed receptor on the surface of each of tumor cells. The immune cell modified with the targeting ligand disclosed in the invention can be specifically connected in a targeted way with the tumor cells, and then the modified immune cell generates and secretes a great number of cytokines which perforate the surfaces of the tumor cells or engulf and lyse tumor cells, so the immune cell modified with the targeting ligand achieves an effect of specifically killing and damaging tumor cells.

(46) The above embodiments are merely preferred embodiments of the invention. It should be noted that, various improvements and modifications made by those ordinarily skilled within the concept of the invention should all fall within the protective scope of the invention.