Gamma delta T cell targeting CD19 / CD20 and application thereof

Abstract

The invention relates to a gamma delta T cell targeting CD19 / CD20 and application thereof, the gamma delta T cell targeting CD19 / CD20 is prepared through non-natural glucose metabolism labeling and biological orthogonal reaction, and the cell has a specific targeting function on CD19 and / or CD20, so that a targeted killing effect on peripheral blood B cells of a patient with systemic lupus erythematosus can be realized.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering and medical technology, and in particular to a CD19 / CD20-targeted γδT cell and an application thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that exhibits high heterogeneity in tissues and organs, and the cause of its onset remains unclear. B cells and plasma cells (effector B cells) play an important role in the pathogenesis of SLE. B cells react with self-antigens to produce autoantibodies. Excessive production of autoantibodies leads to an inflammatory cascade and a large-scale immune response, ultimately causing organ damage in patients. In addition to secreting autoantibodies, B cells also secrete cytokines that promote inflammation, thereby inducing the activation of the body's immune cells and causing the inflammatory response to continue to intensify.

[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 the treatment of SLE 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 in SLE. In addition, γδT cells can secrete anti-inflammatory cytokines, such as IL-10, which help regulate the immune response of SLE and reduce the occurrence of inflammation. These functions make γδT cells a potential tool in the treatment of SLE, especially in the development of new cell therapy technologies. However, technologies involving γδT targeted cell therapy remain to be developed. Summary of the invention

[0004] The purpose of the present invention is to provide a CD19 / CD20-targeted γδT cell and a preparation method and application thereof. The cell has a specific targeting function for CD19 and / or CD20, thereby being able to achieve a targeted killing effect on peripheral blood B cells of patients with systemic lupus erythematosus.

[0005] To this end, in a first aspect, the present invention provides a CD19 / CD20-targeted γδT cell, 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 CD19 and / or CD20; 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] 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.

[0010] By applying the above-mentioned non-natural sugar molecules, γδT cells can be efficiently labeled with the first active group, which is further beneficial for connecting them to CD19 and / or CD20 targeting groups, so that γδT cells have specific targeting functions for CD19 and / or CD20, thereby achieving targeted killing of peripheral blood B cells in patients with systemic lupus erythematosus.

[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] 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.

[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 includes at least one selected from the group consisting of: azide, alkynyl, trans-cyclooctene, triazine, tetrazine, hydrazide, cyclopropene, isocyanide, 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] R7 is selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

[0031] In some embodiments, R7 is 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 CD19 and / or CD20.

[0035] In some embodiments, the targeting group includes at least one selected from the following groups: immunoglobulin, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single region antibody that specifically binds to CD19 and / or CD20, or a monoclonal antibody or polyclonal antibody made from the above antibodies.

[0036] The second aspect of the present invention provides a method for preparing the CD19 / CD20-targeted γδT cells according to the first aspect of the present invention, comprising: connecting a γδT cell modified with a first active group with a targeting group modified with a second active group through a bioorthogonal reaction to obtain the CD19 / CD20-targeted γδT cell; the targeting group targets CD19 and / or CD20; 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] 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.

[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 CD19 / CD20-targeted γδT cells in the preparation of a product for treating systemic lupus erythematosus.

[0045] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0046] (1) The present invention provides CD19 / CD20-targeted γδT cells and a preparation method thereof. Through a corresponding antibody coupling method, a group with a targeting function for CD19 and / or CD20 can be quickly and efficiently modified on the surface of γδT cells, thereby achieving a targeted killing effect on B cells of SLE patients.

[0047] (2) The method for preparing CD19 / CD20-targeted γδT cells provided by the present invention has the advantages of simple operation and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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:

[0049] Figure 1 :Flow chart for preparing CD19 / CD20-targeted γδT cells and schematic diagram of their application;

[0050] Figure 2 : Characterization results of peripheral blood B cells of SLE patients prepared in Example 1;

[0051] Figure 3 : Characterization results of CD19-targeting γδT cells prepared in Example 2;

[0052] Figure 4 : Characterization results of CD20-targeted γδT cells prepared in Example 2;

[0053] Figure 5 :Statistical results of the killing effect of γδT cells targeting CD19 on peripheral blood B cells in SLE;

[0054] Figure 6 :Statistical results of the killing effect of γδT cells targeting CD20 on peripheral blood B cells in SLE. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] The present invention couples a group with a targeting function to CD19 and / or CD20 to γδT cells, so that the group can obtain the function of targeting CD19 / CD20 while retaining the tumor recognition and killing functions, thereby achieving the targeted killing effect of B cells in SLE patients. In order to efficiently label CD19 and / or CD20 targeting groups 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, and high stability.

[0058] Unless otherwise specified, the reagents used in the examples of the present invention can be obtained through conventional commercial routes. In the examples of the present invention, the non-natural sugar molecule shown in formula 3-2-1 can be synthesized with reference to the document Chem. Eur. J. 2023, 29, e202203054.

[0059] Example 1 Preparation and characterization of peripheral blood B cells from SLE patients

[0060] 1. Isolation of peripheral blood mononuclear cells (PBMC) from SLE patients

[0061] (1) Sampling peripheral blood samples from SLE patients: 15 ml of human peripheral blood lymphocyte separation solution was added to a 50 ml conical bottom centrifuge tube. 15 ml of diluted peripheral blood cells was slowly added along the wall of the test tube to an equal volume of lymphocyte separation solution, and centrifuged at 700 g for 20 min.

[0062] (2) After centrifugation, take the middle white flocculent lymphocyte layer and transfer the lymphocytes to a new 50 ml centrifuge tube. Add physiological saline to a total volume of 40 ml and centrifuge at 500 g for 10 min to obtain PBMC cells from SLE patients.

[0063] 2. Preparation and Characterization of B Cells from SLE Patients

[0064] Using EasySep TM B cells were isolated from PBMC of SLE patients using B cell isolation kit. The obtained B cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S. The isolated B cells were characterized using CD19 / CD20 flow cytometry antibodies. The characterization results are shown in Figure 2 shown.

[0065] Example 2 Preparation and characterization of CD19 / CD20-targeted γδT cells

[0066] 1. Preparation and culture of γδT cells

[0067] (1) PBMCs were extracted from healthy human peripheral blood using density gradient centrifugation and the concentration was 2 × 10 6 The cells were inoculated at a density of 100 / ml in γδT cell activation medium (Table 1) and cultured in a cell culture incubator under the following conditions: 37°C, 5% CO2.

[0068] Table 1. γδ T cell activation medium

[0069]

[0070]

[0071] (2) Subculture every 2-3 days, and control the subculture density at 1-2×10 6 The cells were expanded and cultured for about 12 days to prepare γδT cells.

[0072] 2. Preparation of γδT cells targeting CD19 and CD20

[0073]

[0074] (1) Using 100 μM of the non-natural sugar molecule shown in formula 3-2-1 to label γδT cells, the azide group was modified. The specific steps are as follows:

[0075] Take the γδT cells prepared in step 1, add 100 μM non-natural sugar molecules and co-culture for 24 hours; then collect the cells by centrifugation at 400g for 3 minutes, and wash once with PBS to obtain γδT cells labeled with azide groups, namely γδT-N3.

[0076] (2) CD19 antibody Anti-Human CD19 Monoclonal Antibody (Tafbio, BiointronBiological Inc.) and CD20 antibody Rituximab (Rituximab Injection, Roche Diagnostics GmbH) were labeled with DBCO-PEG4-NHS at a concentration ratio of 1:5 and reacted at 30°C for 1 h. The reaction system was then passed through a desalting column to remove unreacted DBCO-PEG4-NHS to obtain CD19-DBCO and CD20-DBCO.

[0077] CD19-DBCO and CD20-DBCO were co-incubated with γδT-N3 cells labeled with azide groups at 37°C for 30 minutes to obtain γδT cells targeting CD19 and γδT cells targeting CD20.

[0078] After labeling with fluorescent secondary antibodies, the cells were characterized by flow cytometry. The results are shown in Figure 3 and Figure 4 As shown, the horizontal axis is the fluorescence intensity and the vertical axis is the number of cells, indicating that the cell surface is successfully labeled with CD19 or CD20, and the labeling efficiency is high.

[0079] Example 3 Cytotoxic effect of CD19 / CD20-targeted γδT cells on peripheral blood B cells in SLE

[0080] (1) Count the peripheral blood B cells of SLE patients prepared in Example 1, at a rate of 1×10 4 100 μL of culture medium was added to a 96-well plate. Peripheral blood B cells from SLE patients were co-cultured with γδT cells targeting CD19, γδT cells targeting CD20, and γδT cells, respectively, and a control group that only entered the culture medium was set up.

[0081] (2) Count the CD19-targeting γδT cells prepared in Example 2, and calculate 1×10 4 / 50μL, 2×10 4 / 50μL, 4×10 4 Add 50 μL of culture medium to the 96-well plate in step (1) and mix the cells thoroughly.

[0082] (3) Count the CD20-targeted γδT cells prepared in Example 2, and calculate 1×10 4 / 50μL, 2×10 4 / 50μL, 4×10 4 Add 50 μL of culture medium to the 96-well plate in step (1) and mix the cells thoroughly.

[0083] (4) Count the γδT cells prepared in step 1 of Example 2, and calculate 1×10 per well. 4 / 50μL, 2×10 4 / 50μL, 4×10 4 Add 50 μL of culture medium to the 96-well plate in step (1) and mix the cells thoroughly.

[0084] (5) Place in an incubator and incubate for 4 hours;

[0085] (6) Add 10 μL CCK8 to each well and incubate in an incubator for 1 hour;

[0086] (7) Measure the absorbance value at 450 nm using an ELISA reader;

[0087] (8) After deducting the effector cell background, the absorbance ratio with the control group is calculated to obtain the number of surviving target cells. The statistical results are as follows: Figure 5 and Figure 6 According to the results, compared with unlabeled γδT cells, the CD19 / CD20-targeted γδT cells provided by the present invention have a significantly better killing effect on peripheral blood B cells of SLE patients.

[0088] 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 CD19 / CD20, characterized in that: The method 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 CD19 and / or CD20; 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 CD19 / CD20-targeted γδT cell 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 CD19 / CD20-targeting γδT cell according to claim 1, characterized in that 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 CD19 / CD20-targeted γδT cell according to claim 1, characterized in that The non-natural sugar molecule comprises at least one selected from the following group:

5. The CD19 / CD20-targeted γδT cell according to claim 1, 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.

6. The CD19 / CD20-targeted γδT cell according to claim 1, characterized in that 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.

7. The CD19 / CD20-targeted γδT cell according to any one of claims 1 to 6, characterized in that: The targeting group includes an antibody or a fragment thereof that specifically binds to CD19 and / or CD20; Preferably, the targeting group comprises 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 CD19 and / or CD20, or a monoclonal antibody or a polyclonal antibody prepared from the above antibodies.

8. The method for preparing CD19 / CD20-targeted γδT cells according to any one of claims 1 to 7, characterized in that: include: The γδT cells 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 cells targeting CD19 / CD20; the targeting group targets CD19 and / or CD20; 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.

9. The preparation method according to claim 8, 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.

10. Use of the CD19 / CD20-targeted γδT cells according to any one of claims 1 to 7 in the preparation of a product for treating systemic lupus erythematosus.

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