Targeting Trop-2 gamma delta T cell as well as preparation method and application thereof

Abstract

The invention relates to a Trop-2 targeting gamma delta T cell and a preparation method and application thereof, the gamma delta T cell is modified with a first active group through non-natural glucose metabolism labeling, and then the first active group and a Trop-2 targeting group modified with a second active group are subjected to a biological orthogonal reaction to obtain the Trop-2 targeting gamma delta T cell. The cell has targeting and selective killing effects on Trop-2 positive tumor cells, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering and medical technology, and in particular to a γδT cell targeting Trop-2 and a preparation method and application thereof. Background Art

[0002] Trophoblast cell surface antigen 2 (Trop-2) is a cell surface glycoprotein encoded by the TACSTD2 gene and is a single-pass transmembrane protein. Trop-2 plays an important role in the development of embryonic organs, but at the same time, it is also an oncogene associated with the occurrence, invasion and metastasis of malignant tumors. In recent years, studies on the molecular mechanism of Trop-2 in malignant tumors have been widely carried out. Trop-2 is upregulated in most tumor cells and drives tumor growth through similar mechanisms in different cell types and species. Some in vivo experiments have shown that Trop-2 quantitatively stimulates tumor growth, and its overexpression can significantly promote tumor progression, such as increased tumor invasiveness and malignancy. Trop-2 is also considered a marker of poor prognosis in a variety of solid tumors, such as breast cancer, ovarian cancer, gastric cancer, colon cancer, lung cancer, papillary thyroid carcinoma, prostate cancer, hepatocellular carcinoma and oral squamous cell carcinoma. In recent years, drug development targeting Trop-2 targets has been continuously carried out, and certain research progress has been made.

[0003] T cells are divided into two categories according to their TCRs: αβT cells (such as CD4, CD8, etc.) and γδT cells. Although γδT cells account for only 0.5%-5% of T cells, they play a vital role in treatment due to their unique immunological properties. γδT cells do not rely on the major histocompatibility complex (MHC) to recognize antigens, so they can respond quickly and widely recognize a variety of abnormal cells. However, technologies related to γδT targeted cell therapy remain to be developed. Summary of the invention

[0004] The purpose of the present invention is to provide a γδT cell targeting Trop-2 and a preparation method and application thereof. The γδT cell has the function of specifically targeting Trop-2, thereby playing a specific and efficient killing effect on Trop-2 positive tumor cells.

[0005] To this end, in a first aspect, the present invention provides a γδT cell targeting Trop-2, which is obtained by connecting a γδT cell modified with a first active group and a targeting group modified with a second active group through a bioorthogonal reaction; the targeting group targets Trop-2; the second active group is suitable for bioorthogonal reaction with the first active group;

[0006] Wherein, the γδT cells are metabolically labeled by non-natural sugar molecules to obtain the γδT cells modified with the first active group; the non-natural sugar molecules include at least one selected from Formula 1 to Formula 6:

[0007]

[0008] Wherein, X is the first active group;

[0009] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to substitution by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 16 alkyl.

[0010] By applying the above-mentioned non-natural sugar molecules, γδT cells can be efficiently labeled with the first active group, which is further conducive to its connection with the Trop-2 targeting group, so that the γδT cells have a specific targeting function for Trop-2, thereby achieving a targeted killing effect on Trop-2 positive tumor cells.

[0011] In some embodiments, the non-natural sugar molecule includes at least one selected from the group consisting of:

[0012]

[0013] Wherein, X is the first active group;

[0014] R 1 , R 2 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 16 alkyl.

[0015] In some embodiments, the non-natural sugar molecule includes at least one selected from the group consisting of:

[0016]

[0017]

[0018] Wherein, X is the first active group.

[0019] In some embodiments, the bioorthogonal reaction comprises at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitization, and glycosylation.

[0020] In some embodiments, the first reactive group comprises at least one selected from the group consisting of azide, alkynyl, trans-cyclooctene, triazine, tetrazine, hydrazide, cyclopropene, isocyanate, and cyclooctyne.

[0021] In some embodiments, the non-natural sugar molecule includes at least one selected from the group consisting of:

[0022]

[0023] In some embodiments, the γδ T cells are human γδ T cells.

[0024] In some embodiments, the γδT cells include at least one of the following subtypes: Vγ9Vδ2T cell subtype, Vδ1T cell subtype.

[0025] In some embodiments, the second reactive group is modified toward the targeting group by a linking compound; the linking compound has both a group that can be covalently linked to a thiol group and the second reactive group.

[0026] In some embodiments, the group that can be covalently linked to a thiol group is a succinimide active ester group, a maleimide group, or a maleic anhydride group.

[0027] In some embodiments, the linking compound comprises a molecule selected from Formula L1 and Formula L2:

[0028]

[0029] Wherein, Y is the second active group;

[0030] R 7 Selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

[0031] In some embodiments, R 7 Selected from C 4～20 Alkylene or C 4～20 Alkylene ether group.

[0032] In some embodiments, the linker compound is DBCO-PEG4-NHS or DBCO-PEG4-MAL;

[0033]

[0034] In some embodiments, the targeting group comprises an antibody or fragment thereof that specifically binds to Trop-2. In some embodiments, the targeting group comprises at least one selected from the group consisting of an immunoglobulin, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single region antibody that specifically binds to Trop-2, or a monoclonal antibody or polyclonal antibody made from the above antibodies.

[0035] In some embodiments, the antibody that specifically binds Trop-2 comprises Sacituzumab.

[0036] The second aspect of the present invention provides a method for preparing the γδT cells targeting Trop-2 according to the first aspect of the present invention, comprising: connecting the γδT cells modified with a first active group and the targeting group modified with a second active group through a bioorthogonal reaction to obtain the γδT cells targeting Trop-2; the targeting group targets Trop-2; the second active group is suitable for undergoing a bioorthogonal reaction with the first active group;

[0037] Wherein, the γδT cells are metabolically labeled by non-natural sugar molecules to obtain the γδT cells modified with the first active group; the non-natural sugar molecules include at least one selected from Formula 1 to Formula 6:

[0038]

[0039] Wherein, X is the first active group;

[0040] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 16 alkyl.

[0041] In some embodiments, the metabolic labeling step comprises: culturing the γδT cells using a culture medium containing the non-natural sugar molecule.

[0042] In some embodiments, the concentration of the non-natural sugar molecule in the culture medium is 10 to 2000 μM; for example, it can be about 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, etc.

[0043] In some embodiments, the culturing time is 2 to 48 hours; for example, it can be about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, etc.

[0044] The third aspect of the present invention provides the use of the Trop-2-targeted γδT cells in the preparation of products for treating Trop-2-positive diseases.

[0045] In some embodiments, the Trop-2 positive disease comprises a Trop-2 positive tumor.

[0046] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0047] (1) The present invention provides γδT cells targeting Trop-2 and a preparation method thereof. Through a corresponding antibody coupling method, a group with a targeting function to Trop-2 can be quickly and efficiently modified on the surface of γδT cells to achieve a targeted killing effect on Trop-2 positive tumor cells.

[0048] (2) The method for preparing γδT cells targeting Trop-2 provided by the present invention has the advantages of simple operation and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0050] Figure 1 :Flow chart for preparing γδT cells targeting Trop-2 and schematic diagram of their application;

[0051] Figure 2 : The proportion of γδT cells in samples after culturing PBMC isolated from healthy subjects;

[0052] Figure 3: γδT cells were labeled with azide groups using non-natural sugar molecules, and the labeling of azide groups was characterized by laser confocal imaging;

[0053] Figure 4 : γδT cells were labeled with azide groups using non-natural sugar molecules, and the labeling of azide groups was characterized by flow cytometry;

[0054] Figure 5 : Characterization results of DBCO group labeled Sacituzumab antibody; Among them, Figure 5 A is the UV absorption spectrum of Sacituzumab antibody; Figure 5 B is the UV absorption spectrum of the DBCO-labeled Sacituzumab antibody, and the DBCO group has an absorption peak at 310 nm; Figure 5 C is the number of DBCO groups labeled on each Sacituzumab antibody molecule calculated based on UV absorption;

[0055] Figure 6 :The prepared γδT-Sacituzumab cells were characterized by laser confocal imaging;

[0056] Figure 7 :The prepared γδT-Sacituzumab cells were characterized by flow cytometry;

[0057] Figure 8 :γδT-Sacituzumab cell-specific killing effect on Trop-2 positive pancreatic cancer cell CFPAC-1;

[0058] Fig. 9 : The killing effect of γδT-Sacituzumab on Trop-2 positive lung cancer cells NCI-H1975, HCC827, NCI-H1650, and PC-9 (D). DETAILED DESCRIPTION

[0059] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0060] In the prior art, immune cell therapy is mainly based on T lymphocytes, among which the T cell surface receptor (TCR) molecules of most (about 95%) mature T cells are composed of two heterodimeric peptide chains, α chain and β chain; a small part is composed of γ and δ chains, namely γδT cells. γδT cells are T cells that perform innate immune functions. They can kill cancer cells and tumor stem cells, and they are immune cells that can recognize cancer antigens, and have strong lethality.

[0061] The present invention couples a group with a targeting function to Trop-2 to γδT cells, so that the group can obtain the function of targeting Trop-2 while retaining the tumor recognition and killing functions, thereby achieving the targeted killing effect of Trop-2 positive tumor cells. In order to efficiently label the Trop-2 targeting group to γδT cells, the present invention provides a non-natural sugar molecule shown in formulas 1 to 6, which can be labeled on the surface of γδT cells by metabolic labeling. The first active group with bioorthogonal reactivity has the advantages of high labeling efficiency, rapidity, high stability, etc.

[0062] Unless otherwise specified, the reagents used in the examples of the present invention can be obtained through conventional commercial routes.

[0063] Example 1 Preparation and characterization of γδT cells

[0064] 1. Extraction of peripheral blood mononuclear cells (PBMC)

[0065] Human peripheral blood was drawn and human PBMCs were separated using lymphocyte separation medium.

[0066] 2. PBMC-Initiated Expansion of γδT Cells

[0067] The medium described in Table 1 was used at 2×10 6 Resuspend γδT cells in 100 cells / mL and culture in a 37°C, 5% carbon dioxide incubator. Culture statically for the first three days, and then culture and passage the cells normally after three days. The flow cytometry characterization results before and after PBMC culture are as follows: Figure 2 As shown, the characterization results show that the purity of γδT cells can reach more than 95% on the ninth day of culture.

[0068] Table 1. γδ T cell activation medium

[0069]

[0070] Example 2 Non-natural sugar labeling of γδT cells

[0071]

[0072] The non-natural sugar molecule shown in formula 3-2-1 is used to label γδT cells and the azide group is modified. The specific steps are as follows:

[0073] The purity-identified γδT cells of Example 1 were taken, 100 μM non-natural sugar molecules were added and co-cultured for 24 h; then the cells were collected by centrifugation at 400 g for 3 min, and washed once with PBS to obtain γδT cells labeled with azide groups, i.e., γδT-N 3 Take 5×10 4 The cells were verified by incubating with 100 μM DBCO-PEG4-Biotin at room temperature for 10 minutes. After the incubation, the cells were washed with PBS. Then, the cells were incubated with 2 μg / ml Streptavidin-Alexa Fluor 488 at 4°C for 15 minutes. After the incubation, the cells were washed with PBS. Then, laser confocal imaging and flow cytometry were used to detect AF488 fluorescence to characterize the cell azide labeling. The results of laser confocal imaging are shown in Figure 2. Figure 3 As shown in the figure, the red color indicates the non-natural sugar marker; the flow cytometry detection results are shown in Figure 4 shown. Figure 3 and Figure 4 The results showed that there were azide groups on the cell surface, with extremely high labeling efficiency.

[0074] Example 3 Labeling Sacituzumab with DBCO Group

[0075] DBCO (dibenzocyclooctene) can be used for the azide-alkyne cycloaddition reaction without copper ion catalysis. It is used as the second active group to modify the Sacituzumab antibody with Trop-2 targeting function. The specific steps are as follows:

[0076] The Sacituzumab antibody was labeled with DBCO-PEG4-NHS at a concentration ratio of 1:5 and reacted at 30°C for 1 hour; then the reaction system was passed through a desalting column to remove unreacted DBCO-PEG4-NHS to obtain a Sacituzumab antibody labeled with DBCO, namely Sacituzumab-DBCO. Figure 5 The characterization results of bioorthogonal DBCO group labeled antibodies are shown, wherein: Figure 5 A is the UV absorption spectrum of Sacituzumab antibody; Figure 5 B is the UV absorption spectrum of the DBCO-labeled Sacituzumab antibody, and the DBCO group has an absorption peak at 310 nm; Figure 5 C is the number of DBCO groups labeled on each sacituzumab antibody molecule calculated based on UV absorption.

[0077] Example 4 γδ T cells targeting Trop-2

[0078] The γδT cells modified with an azide group prepared in Example 2 and the Sacituzumab-DBCO prepared in Example 3 were co-incubated at 37°C for 30 min, and then the cells were collected by centrifugation at 300g for 5 min, and the cells were washed once with PBS to obtain γδT cells that can target Trop-2, namely γδT-Sacituzumab.

[0079] The prepared γδT-Sacituzumab was characterized and labeled with PE-anti-human Fc (Biolegend, Catalog No.: 410707), and then fluorescence was detected by laser confocal imaging and flow cytometry. Figure 6 This is the confocal microscope imaging result. The green fluorescence indicates the cells labeled with Sacituzumab. Figure 7 is the detection result of flow cytometry. Figure 6 and Figure 7 The results showed that γδT cells labeled with Sacituzumab were successfully prepared.

[0080] The killing effect of γδT-Sacituzumab on Trop-2 positive tumor cells was verified by the following experiments.

[0081] Trop-2-negative AsPC-1 cell line and Trop-2-positive CFPAC-1 cell line were digested and plated, and 5000 cells were added to each well of a 96-well plate at 37°C and 5% CO. 2 Culture overnight. Then, γδT-Sacituzumab cells and γδT cells were co-incubated with the above cells at an effector-target ratio of 0.5:1 and 1:1, respectively, and incubated at 37°C for 3 hours. After the incubation, the LDH detection kit was used to detect the cell killing effect. The results are as follows Figure 8 As shown, γδT cells modified with Sacituzumab antibody exhibited specific and efficient killing effects on Trop-2-positive pancreatic cancer cells CFPAC-1.

[0082] Furthermore, the killing effect of γδT-Sacituzumab on other Trop-2 positive tumor cells was verified. The cells used in the experiment included: Trop-2 positive lung cancer cells NCI-H1975, HCC827, NCI-H1650, PC-9, and the experiment was carried out in the same way as above. The results are shown in Fig. 9 As shown, it can be seen that the γδT cells modified with the Sacituzumab antibody provided by the present invention have a highly effective killing effect on a variety of Trop-2 positive tumor cells.

[0083] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A γδT cell targeting Trop-2, characterized in that: The γδT cells targeting Trop-2 are obtained by connecting γδT cells modified with a first active group and a targeting group modified with a second active group through a bioorthogonal reaction; the targeting group targets Trop-2; and the second active group is suitable for bioorthogonal reaction with the first active group; Wherein, the γδT cells are metabolically labeled by non-natural sugar molecules to obtain the γδT cells modified with the first active group; the non-natural sugar molecules include at least one selected from Formula 1 to Formula 6: Wherein, X is the first active group; R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 16 alkyl.

2. The γδT cell targeting Trop-2 according to claim 1, characterized in that The non-natural sugar molecule comprises at least one selected from the following group: Wherein, X is the first active group; R1 and R2 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 16 alkyl; Preferably, the non-natural sugar molecule comprises at least one selected from the following group: Wherein, X is the first active group.

3. The Trop-2-targeting γδT cell according to claim 1, wherein The bioorthogonal reaction comprises at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitization, and glycosylation; Preferably, the first reactive group comprises at least one selected from the group consisting of azide, alkynyl, trans-cyclooctene, triazine, tetrazine, hydrazide, cyclopropene, isocyanate, and cyclooctyne.

4. The Trop-2-targeting γδT cell according to any one of claims 1 to 3, characterized in that The γδT cells are human γδT cells; Preferably, the γδT cells include at least one of the following subtypes: Vγ9Vδ2T cell subtype and Vδ1T cell subtype.

5. The Trop-2-targeting γδT cell according to claim 1, wherein Modifying the second reactive group to the targeting group by a linking compound; the linking compound simultaneously has a group covalently linkable to a thiol group and the second reactive group; Preferably, the group that can be covalently linked to the thiol group is a succinimide active ester group, a maleimide group or a maleic anhydride group; Preferably, the linking compound comprises a molecule selected from Formula L1 and Formula L2: Wherein, Y is the second active group; R7 is selected from C 1～20 Alkylene or C 1～20 Alkylene ether group; Preferably, the linking compound is DBCO-PEG4-NHS or DBCO-PEG4-MAL.

6. The Trop-2-targeting γδT cell according to any one of claims 1 to 5, characterized in that The targeting group includes an antibody or a fragment thereof that specifically binds to Trop-2; In some embodiments, the targeting group includes at least one selected from the following group: an immunoglobulin, Fab, Fab', F(ab')2, Fv, scFv, a bispecific antibody, a multispecific antibody, a single domain antibody or a single region antibody that specifically binds to Trop-2, or a monoclonal antibody or a polyclonal antibody prepared from the above antibodies.

7. The method for preparing γδT cells targeting Trop-2 according to any one of claims 1 to 6, characterized in that: include: The γδT cell modified with a first active group and a targeting group modified with a second active group are connected through a bioorthogonal reaction to obtain the γδT cell targeting Trop-2; the targeting group targets Trop-2; the second active group is suitable for bioorthogonal reaction with the first active group; Wherein, the γδT cells are metabolically labeled by non-natural sugar molecules to obtain the γδT cells modified with the first active group; the non-natural sugar molecules include at least one selected from Formula 1 to Formula 6: Wherein, X is the first active group; R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 16 alkyl.

8. The preparation method according to claim 7, characterized in that: The metabolic labeling step comprises: culturing the γδT cells using a culture medium containing the non-natural sugar molecule; Preferably, in the culture medium, the concentration of the non-natural sugar molecule is 10 to 2000 μM; Preferably, the culturing time is 2 to 48 hours.

9. Use of the Trop-2-targeted γδT cells according to any one of claims 1 to 6 in the preparation of products for treating Trop-2-positive diseases.

10. The use according to claim 10, characterized in that The Trop-2 positive disease includes Trop-2 positive tumors.

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