ADAM17 in the preparation of therapeutic effector CD8 + Application in medicine for T cell differentiation-related diseases

Abstract

The present invention belongs to the technical field of anti-tumor drug research, and specifically discloses a method for preparing a therapeutic effector CD8 + The application of drugs in T cell differentiation related diseases. Experiments have shown that in mouse tumor models, adoptive specific modification of ADAM17 CD8 + T cells compared with wild-type CD8 + T cells can significantly inhibit tumor growth, and the exhaustion of CD8 + The proportion of T cells decreased. + Modification of ADAM17 in T cells can effectively increase CD8 + T cells can clear bacteria and enhance the ability to resist infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug research, and specifically discloses a method for preparing a therapeutic effector CD8 + Use of the invention in medicine for T cell differentiation-related diseases. Background Art

[0002] CD8 + T cells play a vital role in anti-tumor activities, but it is currently known that CD8 + Most of the regulation of T cells is at the transcription and translation level and is an intracellular molecule, which greatly limits the clinical development of immunotherapy drugs. ADAM17 is a member of the metalloproteinase family and a transmembrane protein. It plays a key role in a variety of diseases by cleaving cell surface protein molecules. The inflammatory cytokine TNF-α needs to be cleaved by ADAM17 before it can become a soluble TNF-α with pro-inflammatory function. The IL-6 receptor IL-6R on the membrane surface is cleaved by AMDA17 to form a soluble IL-6R, which combines with IL-6 to form a complex, triggering a strong inflammatory response in cells, which is called the trans signaling pathway of IL-6. It can be seen that ADAM17 is an important target for the development of therapeutic drugs, but ADAM17 has a role in adaptive immunity, especially CD8 + The role of T cells in differentiation and immune response is still unclear. Summary of the Invention

[0003] In view of the above technical problems, the present invention provides the following technical problems:

[0004] The present invention provides a method for preparing a therapeutic effector CD8 + Use in medicine for T cell differentiation-related diseases.

[0005] Preferably, the effector CD8 + Diseases related to T cell differentiation include tumors or Listeria infection.

[0006] Preferably, the tumor is cutaneous melanoma.

[0007] Preferably, the ADAM17 is combined with pharmaceutically acceptable excipients or carriers to prepare a drug.

[0008] Compared with the prior art, the beneficial effects of the present invention are:

[0009] 1. The present invention provides ADAM17 in the preparation of therapeutic effector CD8 + In a mouse tumor model, adoptive delivery of ADAM17-modified CD8 + T cells compared with wild-type CD8 +T cells can significantly inhibit tumor growth, and the exhaustion of CD8 + The proportion of T cells is reduced, so ADAM17 can act as an effector CD8 + Potential therapeutic targets for T cell-related diseases.

[0010] 2. It has been verified through experiments that in CD8 + Modification of ADAM17 in T cells can effectively increase CD8 + T cells can clear bacteria and enhance the ability to resist infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a schematic diagram of ADAM17-specific knockout transgenic mice in the OT-I background;

[0012] Figure 2 Identification of T cell-specific knockout ADAM17 transgenic mice; A, agarose electrophoresis diagram of transgenic mouse gene identification, B, qPCR detection of ADAM17 in CD8 + T cell knockout efficiency; *** P≤0.001;

[0013] Figure 3 ADAM17 modifies CD8 + The ability of T cells to clear Listeria; A. Bacterial content in the spleen, B. Bacterial content in the liver; C. Clearance of bacteria in the spleen; D. Clearance of bacteria in the liver; Compared with the control group, *** P≤0.001;

[0014] Figure 4 ADAM17 modifies CD8 + Anti-tumor ability of T cells; A, tumor growth curve, B, mouse survival curve; compared with the control group, ** P≤0.01; DETAILED DESCRIPTION

[0015] The technical scheme of the present application will be described in further detail below in conjunction with specific preparation examples and examples. The following preparation examples and examples are merely illustrative and explain the present application, and should not be construed as limiting the scope of protection of the present application. Those skilled in the art may make non-essential improvements and adjustments to the embodiment according to the foregoing invention, which still fall within the scope of protection of the present application. Unless otherwise indicated, the raw materials, reagents (for example, organic solvents, non-polar solvents, kinases, substrates, antibodies, buffer solutions, reaction solutions, etc.) used in the following preparation examples and examples are all commercially available products, or can be prepared or formulated by known methods or reagent instructions.

[0016] The present invention provides ADAM17 in the preparation of therapeutic effector CD8+ Use of the invention in medicine for T cell differentiation-related diseases.

[0017] Example 1

[0018] ADAM17 transgenic mice (purchased from Jackson Laboratory) were hybridized with T cell tool mice (Lck-cre) to obtain mice with ADAM17 specifically deleted in T cells. These mice were then hybridized with OT-I mice to obtain transgenic mice with ADAM17 specifically knocked out in T cells on an OT-I background. The specific process is as follows Figure 1 As shown. T cell-specific knockout ADAM17 transgenic mice were identified and CD8 + T cells, qPCR detection of ADAM17 in CD8 + T cell knockout efficiency.

[0019] turn out( Figure 2 ), in transgenic mouse T cells, a loxp site was specifically inserted into the ADAM17 gene, so that the expression of ADAM17 could be knocked out by the T cell tool Lck-cre. + The ADAM17 gene was completely knocked out in T cells, indicating that CD8 + ADAM17-modified CD8 T cells + T cells.

[0020] Example 2

[0021] ADAM17-specifically modified CD8 + T cells enhance resistance to bacterial infection

[0022] Adoptive wild-type CD8 + T cells and ADAM17-modified CD8 T cells isolated in Example 1 + T cells, injected at a volume of 2 × 10 5 cells / mouse, and then the mice were infected with 1×10 6 CFU / mouse Listeria to obtain Listeria infection model. Detect CD8 + T cell clearance rate of Listeria monocytogenes.

[0023] The results show that ( Figure 3 ), compared with wild-type CD8 + T cells, adoptively transferred ADAM17-modified CD8 + Mice with CD8 T cells are more efficient in clearing bacteria. +Modification of ADAM17 in T cells can effectively increase CD8 + T cells can clear bacteria and enhance the ability to resist infection.

[0024] Example 3

[0025] ADAM17-specifically modified CD8 + T cells inhibit skin melanoma growth

[0026] The recipient mice were injected subcutaneously with 1×10 6 cells / skin melanoma tumor cells, and 7 days later, wild-type CD8 + T cells and ADAM17-modified CD8 T cells isolated in Example 1 + T cells, injected at a volume of 2 × 10 5 cells / cells, obtain tumor model, detect CD8 + T cells' ability to suppress cutaneous melanoma growth.

[0027] The results show that ( Figure 4 ), compared with wild-type CD8 + T cells (control group), adoptive ADAM17-modified CD8 + Mice with T cells have a stronger ability to inhibit tumor growth and effectively delay tumor growth. + The proportion of T cells decreased. + Modification of ADAM17 in T cells can effectively increase CD8 + Anti-tumor ability of T cells.

[0028] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. CD8 knockout of ADAM17 gene + T cells in the preparation of therapeutic effector CD8 + The invention relates to a drug for T cell differentiation-related diseases, characterized in that: The effector CD8 + Diseases associated with T cell differentiation include skin melanoma or Listeria infection.

2. The use according to claim 1, characterized in that The drug includes pharmaceutically acceptable excipients or carriers.

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