CN117624379A - An artificial protein and its application - Google Patents

Abstract

The invention belongs to the technical field of artificial proteins, and particularly relates to an artificial protein and application thereof. The artificial protein has unique in-vivo and in-vitro immunocompetence and can meet the requirement of recovering the immune function of a patient; namely, the artificial protein structure can not only identify the exhausted immune cells, but also increase invasiveness and restore functions; and the clinical application of the composition can enhance the functions of inhibiting tumor growth and controlling virus infection, and has good clinical prospect and wide application range.

Description

An artificial protein and its application

Technical field

The invention belongs to the technical field of artificial proteins, and specifically relates to an artificial protein and its application.

Background technique

The occurrence of tumors is due to genetic mutations during the cell division of the body, and the growth of mutant cells is out of control. If tumor cells cannot be eliminated in a short period of time, continued antigen stimulation will gradually cause functional exhaustion of tumor antigen-specific T cells, forming tolerance to tumors. The immune system will no longer be sensitive to tumor cells, causing further tumor growth and spread, eventually forming cancer. The occurrence of chronic diseases caused by viral infection is also the result of antigen-specific cell failure, and numerous studies have proven this conclusion.

Research also shows that due to continuous antigen stimulation, most tumor-infiltrating lymphocytes (TILs) and anti-viral specific T cells highly express immunosuppressive receptors and are in a state of functional exhaustion, weakening or losing specific recognition. Function with killing antigen-positive target cells. Exhausted immune cells lose the ability to secrete functional cytokines, such as gamma interferon (IFNγ). Even if there are a large number of specific immune cells in the body, exhausted immune cells cannot clear antigen-positive target cells. Phenotypically, immunosuppressive checkpoint receptors commonly expressed by exhausted T cells include programmed death receptor (PD-1), programmed death ligand (PD-L1/L2), and T cell immunoglobulin 3. (Tim-3), cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphocyte activation gene 3 (Lag-3), etc. The activation of these signaling pathways will inhibit the phosphorylation reaction by inducing phosphatase, weakening immune cell activation signals, inhibiting the expansion of immune cells, reducing the function of effector immune cells, promoting T cell apoptosis, and causing immune tolerance. Therefore, activating this type of antigen-specific immune cell population and allowing them to regain their function of killing antigen-positive target cells can eliminate tumor cells or virus-infected cells and ultimately achieve cure.

Antibodies can specifically recognize protein antigens on the surface of target cells. Checkpoint inhibitory antibodies can block immunosuppressive signals on the surface of exhausted cells. For example, PD-1 antibodies (Pembrolizumab and Nivolumab) prevent inhibition by binding to PD-1. The conduction and activation of sexual signals and the restoration of the function of exhausted immune cells have shown clinical effects in the treatment of cancer, especially in some patients who have achieved complete tumor disappearance and long-lasting clinical effects. However, the clinical effect for patients who lack effector cells is not ideal, and the recovery duration of this T cell is short, and the specific immune cells in the patient's body will quickly return to a state of exhaustion. At the same time, simply blocking T cell inhibitory signals will not lead to an increase in the number of immune cells, and patients lacking immune effector cells will basically not have clinical effects, further limiting the clinical therapeutic effect of checkpoint antibodies.

Cytokines have been approved for cancer treatment, with interleukin-2 (IL-2) approved for colorectal cancer treatment in 1992. However, only a small number of patients benefit, and most suffer severe side effects. High doses of IL-2 can cause shock or even death in patients. Finally, clinical trials using IL-15 to treat tumors have not produced significant clinical effects.

CD137 is a TNFR receptor expressed by T cells after stimulation. The CD137-CD137L interaction can cause immune cell expansion and survival, enhance immune cell function, and prevent immune cell apoptosis. Among antigen-stimulating cells, CD137 can promote the function of antigen-presenting cells and enhance the activation of T cells. Experiments have shown that activation of CD137 molecules can increase the activity of antigen-specific immune cells. Animal experiments show that only CD137 ⁺ immune cells can control tumor growth. In recent years, CD137 ⁺ T cells have been further proven to control the progression of tumor patients, and their expression in the tumor microenvironment may be inhibited by PD-1 and reduce their activity. Clinical studies have shown that the expression of CD137 in lymphocytes is positively correlated with the survival of patients with colorectal cancer. Furthermore, in patients with lung cancer, the decrease in CD137 ⁺ cells is inversely related to patient survival. At the same time, the proportion of CD8 ⁺ CD137 ⁺ positive T cells was significantly positively correlated with the survival rate of cancer patients, and a high proportion of CD3 ⁺ CD137 ⁺ PD1 ⁺ immune cells significantly reduced the chance of patient deterioration.

Based on the above evidence, up to now, there is no protein drug in this field that can not only specifically identify exhausted immune cells, but also increase the function of immune cells.

Contents of the invention

The purpose of the present invention is to provide an artificial protein and its application, which can both deplete immune cells and enhance the function of immune cells.

The invention provides an artificial protein, which includes a recognition functional region, an activation functional region and a non-functional amino acid fragment connecting the recognition functional region and the activation functional region; the C-terminal of the recognition functional region is connected to the The N-terminus of the activation domain is connected through a non-functional amino acid fragment;

The recognition functional region includes a PD-1 single-chain antibody, and the amino acid of the PD-1 single-chain antibody is as shown in SEQ ID NO.1, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.1;

The activation domain includes the cytokines IL-2, IL-7, IL-15 or IL-21.

Preferably, the amino acid sequence of the cytokine IL-2 is as shown in SEQ ID NO.2, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.2;

The amino acid sequence of the cytokine IL-7 is shown in SEQ ID NO.3, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.3;

The amino acid sequence of the cytokine IL-15 is as shown in SEQ ID NO.4, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.4;

The amino acid sequence of the cytokine IL-21 is shown in SEQ ID NO.5, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.5;

Preferably, the amino acid sequence of the non-functional amino acid fragment is shown in SEQ ID NO. 6.

Preferably, the amino acid sequence of the artificial protein is shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

The present invention also provides the application of the artificial protein described in the above technical solution in the preparation of drugs for restoring immune cell function.

Preferably, the immune cells include T cells and/or NK cells.

Preferably, the drugs include drugs that simultaneously identify exhausted immune cells and/or enhance the function of immune cells.

Preferably, the drugs include drugs for treating cancer and/or chronic viral infections.

Preferably, the cancer includes one or more of renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, bladder cancer and lung cancer.

The present invention also provides a drug for restoring immune cell function, which drug includes the artificial protein described in the above technical solution.

Beneficial effects:

The invention provides an artificial protein, which includes a recognition functional region, an activation functional region and a non-functional amino acid fragment connecting the recognition functional region and the activation functional region; the C-terminal of the recognition functional region is connected to the The N-terminus of the activation functional region is connected through a non-functional amino acid fragment; the recognition functional region includes a PD-1 single-chain antibody, and the amino acids of the PD-1 single-chain antibody are as shown in SEQ ID NO.1; the activation Functional areas include the cytokines IL-2, IL-7, IL-15 or IL-21. The artificial protein of the present invention has unique immune activity in vivo and in vitro, and can meet the needs of restoring the immune function of patients; that is, the artificial protein structure can not only identify exhausted immune cells, but also increase the invasiveness and restore their functions; and Clinical application can enhance the function of inhibiting tumor growth and controlling viral infection, and has good clinical prospects and a wide range of applications.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below.

Figure 1 is an electrophoresis detection chart of the recombinant fusion artificial protein in Examples 1 to 4;

Figure 2 is a diagram showing the activity and function measurement results of the recombinant fusion artificial protein on human T cells in vitro culture in Example 1 and the control group;

Figure 3 is a diagram showing the activity and function measurement results of the recombinant fusion artificial proteins in Examples 2 to 4 on human T cells cultured in vitro.

Detailed ways

The invention provides an artificial protein, which includes a recognition functional region, an activation functional region and a non-functional amino acid fragment connecting the recognition functional region and the activation functional region; the C-terminus of the recognition functional region is connected to the The N-terminus of the activation domain is connected through a non-functional amino acid fragment;

The recognition functional region includes a PD-1 single-chain antibody, and the amino acid of the PD-1 single-chain antibody is as shown in SEQ ID NO.1, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.1;

The activation domain includes the cytokines IL-2, IL-7, IL-15 or IL-21.

In the present invention, the amino acid sequence shown in SEQ ID NO. 1 is specifically: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGSGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPG QGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS. The recognition functional region of the present invention recognizes the phenotypic receptor of exhausted immune cells, and the phenotypic receptor of exhausted immune cells is the immune checkpoint PD-1 with co-suppressive function. Such receptor genes are overexpressed on the surface of immune cells, and activation of this signaling pathway can lead to immune cell exhaustion and loss of function.

In the present invention, the amino acid sequence of the cytokine IL-2 is preferably as shown in SEQ ID NO.2, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.2; the amino acid sequence shown in SEQ ID NO.2 The specific amino acid sequence shown is: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIS NINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

The amino acid sequence of the cytokine IL-7 of the present invention is preferably as shown in SEQ ID NO.3, or a mutant sequence having the same function as the amino acid shown in SEQ ID NO.3; the amino acid sequence shown in SEQ ID NO.3 The sequence is specifically: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN EFNFFKRHICDANNKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIL LNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH.

The amino acid sequence of the cytokine IL-15 of the present invention is preferably as shown in SEQ ID NO.4, or a mutant sequence having the same function as the amino acid shown in SEQ ID NO.4; the amino acid sequence shown in SEQ ID NO.4 The sequence is specifically: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQS MHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

The amino acid sequence of the cytokine IL-21 of the present invention is preferably as shown in SEQ ID NO.5, or a mutant sequence having the same function as the amino acid shown in SEQ ID NO.5; the amino acid sequence shown in SEQ ID NO.5 The sequence is specifically: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPE DVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQK HRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

The cytokine part of the present invention binds to its receptor expressed on the surface of T cells and NK cells, thereby exerting the function of activating and increasing invasiveness. Cytokines may exist in the form of mutants. Mutants refer to enhancing or weakening their effects on non-target cells through amino acid sequence mutations or just the use of functional region peptides without changing their basic functions. For example, through changes in the amino acid sequences of cytokines, mutants may reduce or increase the activation of T regulatory cells, reduce the effect on non-target cells and tissues and organs, thereby reducing the side effects of clinical application.

In the present invention, the amino acid sequence of the non-functional amino acid fragment is preferably as shown in SEQ ID NO.6, specifically: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The non-functional amino acid fragment of the present invention plays a role in connecting the recognition functional domain protein and the activation functional domain protein.

In the present invention, the amino acid sequence of the artificial protein is shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10. The amino acid sequence shown in SEQ ID NO.7 of the present invention is specifically: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQK; the amino acid sequence shown in SEQ ID NO.8 is specifically: EIVLTQSPATLSLSPGERATLSCRASKGVSTS; the amino acid sequence shown in SEQ ID NO.9 is specifically: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAP; the amino acid sequence shown in SEQ ID NO. 10 is specifically: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSY. In the present invention, the SEQ ID NO.7 consists of the PD-1 single chain antibody shown in SEQ ID NO.1, the non-functional amino acid fragment shown in SEQ ID NO.6, linker "GGGS" and SEQ ID NO. The cytokine IL-2 shown in 2 is sequentially connected; the SEQ ID NO.8 is composed of the PD-1 single chain antibody shown in SEQ ID NO.1, the non-functional amino acid fragment shown in SEQ ID NO.6, The linker "GGGS" is sequentially connected to the cytokine IL-7 shown in SEQ ID NO.3; the SEQ ID NO.9 is composed of the PD-1 single chain antibody shown in SEQ ID NO.1, SEQ ID NO. The non-functional amino acid fragment shown in 6, linker "GGGS" and the cytokine IL-15 shown in SEQ ID NO.4 are sequentially connected; the SEQ ID NO.10 is composed of the PD shown in SEQ ID NO.1 -1 single chain antibody, non-functional amino acid fragment shown in SEQ ID NO.6, linker "GGGS" and cytokine IL-21 shown in SEQ ID NO.5 are sequentially connected.

The recognition functional region in the artificial protein shown in SEQ ID NO. 7 to 10 of the present invention recognizes exhausted immune cells and uses the PD-1 single chain antibody to recognize the receptor PD-1 of the phenotype of exhausted immune cells; activate The functional area uses IL-2, IL-7, IL-15 or IL-21 to activate immune cells, thereby identifying exhausted immune cells and enhancing the function of immune cells.

The present invention also provides the application of the artificial protein described in the above technical solution in the preparation of drugs for restoring immune cell function. In the present invention, the immune cells preferably include T cells and/or NK cells, more preferably T cells or NK cells; the T cells preferably include specific T cells.

The drugs of the present invention preferably include drugs that simultaneously identify exhausted immune cells and/or enhance the function of immune cells, further include drugs that simultaneously identify exhausted immune cells and enhance the function of immune cells, and more preferably include drugs that simultaneously block PD-1 of immune cells. Drugs that express and increase CD137 expression. The enhancement of cell function of the present invention preferably includes increasing the invasion function of immune cells.

The medicaments of the present invention preferably include medicaments for treating cancer and/or chronic viral infections, more preferably medicaments for treating cancer and chronic viral infections. The cancers described in the present invention preferably include one or more of renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, bladder cancer and lung cancer.

The present invention also provides a drug for restoring immune cell function, which drug includes the artificial protein described in the above technical solution. The artificial protein of the present invention is preferably an active ingredient in the medicine. The drugs of the present invention are preferably used alone or in combination with other drugs or treatment methods; the other drugs preferably include chemotherapy drugs, targeted drugs or antibody drugs; the treatment methods preferably include cell therapy or radiotherapy.

In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present invention.

Example 1

The steps for gene construction, production and purification of artificial protein structures are as follows:

1) According to the C-terminal amino acid sequence of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 2 (SEQ ID NO.2), through base synthesis, enzyme digestion and further cloning corresponding to the gene, Non-functional artificially constructed amino acids (SEQ ID NO. 6) and linker "GGGS" connect the C-terminal of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 2 (SEQ ID NO.2). An artificial protein structural amino acid sequence (SEQ ID NO.7) was composed, and the nucleotide sequence encoding the artificial protein structural amino acid sequence (SEQ ID NO.7) was cloned into the pcDNA3.1(-) vector.

2) Transfect the expression vector of the protein structure into Chinese hamster ovary cells (CHO). The transfected cells were cultured in a 37°C, 5% CO ₂ incubator. One week later, the supernatant was taken and further purified through ProteinA affinity chromatography. The final purified protein was the designed recombinant fusion artificial protein.

Example 2

1) According to the C-terminal amino acid sequence of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 7 (SEQ ID NO.3), through base synthesis, enzyme digestion and further cloning corresponding to the gene, through Non-functional artificially constructed amino acids (SEQ ID NO. 6) and linker "GGGS" connect the C-terminal of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 7 (SEQ ID NO.3). An artificial protein structural amino acid sequence (SEQ ID NO. 8) was composed, and the nucleotide sequence encoding the artificial protein structural amino acid sequence (SEQ ID NO. 8) was cloned into the pcDNA3.1(-) vector.

2) Same as Example 1.

Example 3

1) According to the C-terminal amino acid sequence of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 15 (SEQ ID NO.4), through base synthesis, enzyme digestion and further cloning corresponding to the gene, through The non-functional artificially constructed amino acid (SEQ ID NO.6) and the linker "GGGS" connect the C-terminal of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 15 (SEQ ID NO.4). An artificial protein structural amino acid sequence (SEQ ID NO. 9) was composed, and the nucleotide sequence encoding the artificial protein structural amino acid sequence (SEQ ID NO. 8) was cloned into the pcDNA3.1(-) vector.

2) Same as Example 1.

Example 4

1) According to the C-terminal amino acid sequence of the human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 21 (SEQ ID NO.5), through base synthesis, enzyme digestion and further cloning corresponding to the gene, through Non-functional artificially constructed amino acids (SEQ ID NO. 6) and linker "GGGS" connect the C-terminal of human PD-1 single-chain antibody (SEQ ID NO.1) and the N-terminal amino acid sequence of interleukin 21 (SEQ ID NO.5). An artificial protein structural amino acid sequence (SEQ ID NO. 10) was composed, and the nucleotide sequence encoding the artificial protein structural amino acid sequence (SEQ ID NO. 8) was cloned into the pcDNA3.1(-) vector.

2) Same as Example 1.

The recombinant fusion artificial proteins prepared in Examples 1 to 4 were subjected to electrophoresis detection, and the results are shown in Figure 1, in which lanes 1 to 4 in Figure 1 represent the results of the recombinant fusion artificial proteins in Examples 1 to 4 in sequence. Show results. The electrophoresis detection in Figure 1 confirms that the molecular weight of the purified protein structure is approximately 67kD, proving that the recombinant artificial protein structure designed according to the present invention can be produced by CHO cells. Finally, the protein concentration was tested using a spectrophotometer and diluted in PBS for further in vitro and in vivo activity testing and functional studies.

Application example 1

The activity and function of the recombinant fusion artificial proteins in Examples 1 to 4 on human T cells cultured in vitro were measured as follows:

Human peripheral blood was separated and purified into the PBMC fraction using lymphocyte density gradient centrifugation (Ficoll), and then diluted with X-Vivo15 culture medium in a 24-well plate to a cell density of 5×10 ⁶ /mL, and the test protein was added to the final concentration. is 100ng/mL; the test proteins are the recombinant fusion artificial proteins in Examples 1 to 4 respectively. Then place them in a 37°C, 5% _CO2 incubator for 72 hours. After collecting the cells, they are stained with the corresponding flow cytometry antibodies (Biolegend) (the corresponding PD-1 antibody is APC and the CD137 antibody is PE). After washing, Flow cytometry was used for phenotyping and data analysis. The results are shown in Figures 2 to 3. In Figures 2 to 3, the positive rate of PD-1 is shown vertically, and the positive rate of CD137 is shown horizontally. In Figure 2, from left to The Control group and the recombinant fusion artificial protein in Example 1 are shown in sequence on the right, and the recombinant fusion artificial proteins in Examples 2 to 4 are shown in Figure 3 from left to right.

Figures 2 to 3 show that compared with the control without protein group, all four artificial protein structure treatments can significantly increase the expression of CD137 in T cells. The specific positive rate of CD137 in the control group is only 4.06%. , and the positive rates of CD137 in the test group added with the recombinant fusion artificial protein in Examples 1 to 4 were 21.2%, 19.8%, 11.3% and 25.7% respectively. At the same time, compared with the control group without protein (7.64%), the expression of PD-1 was significantly reduced after culture with the four protein structures. The positive rate of PD-1 in the specific control group was as high as 7.64%, while The positive rates of PD-1 in the test groups added with the recombinant fusion artificial proteins in Examples 1 to 4 were only 0.14%, 0.05%, 0.03% and 0.02% respectively. Experiments have proven that the recombinant protein structure designed according to the present invention can not only activate human T cells and increase the expression of CD137 molecules, but can also shield the PD-1+ signal on the cell surface to achieve the activity generated by the designed target protein structure.

It can be seen from the above examples that the artificial protein structure of the present invention can not only identify exhausted immune cells, but also activate and increase the invasiveness of immune cells, and restore the function of immune cells in killing antigen-positive cells. The protein structure has the same functions as the non-protein structure. It can not only activate immune cells, but also shield PD-1 signals and induce immune cells to infiltrate tumors. Since the occurrence and spread of tumors and chronic viral infections are the result of immune cell exhaustion and immune system tolerance, restoring the function of exhausted immune cells and expanding the number of immune cells can enhance the body's ability to resist tumors and chronic viral infections. By analogy, the clinical application of the above protein structure can inhibit tumor growth and control viral infection, and has good clinical prospects and a wide range of applications.

The above-mentioned artificial protein is a bifunctional recombinant protein structure, which can be used alone or in combination with chemotherapy, targeted drugs, antibody drugs, cell therapy and radiotherapy to prepare drugs for the treatment of diseases caused by immune cell failure, such as Drugs to treat cancer and drugs to treat chronic viral infections, among others.

Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without any inventive step. These embodiments All belong to the protection scope of the present invention.

Claims (10)

1. An artificial protein, characterized in that the artificial protein includes a recognition functional region, an activation functional region and a non-functional amino acid fragment connecting the recognition functional region and the activation functional region; the C-terminus of the recognition functional region and The N-terminus of the activation functional region is connected through a non-functional amino acid fragment; The recognition functional region includes a PD-1 single-chain antibody, and the amino acid of the PD-1 single-chain antibody is as shown in SEQ ID NO.1, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.1; The activation domain includes the cytokines IL-2, IL-7, IL-15 or IL-21. 2. The artificial protein according to claim 1, characterized in that the amino acid sequence of the cytokine IL-2 is as shown in SEQ ID NO.2, or a mutation with the same function as the amino acid shown in SEQ ID NO.2. body sequence; The amino acid sequence of the cytokine IL-7 is as shown in SEQ ID NO.3, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.3; The amino acid sequence of the cytokine IL-15 is as shown in SEQ ID NO.4, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.4; The amino acid sequence of the cytokine IL-21 is shown in SEQ ID NO.5, or a mutant sequence with the same function as the amino acid shown in SEQ ID NO.5. 3. The artificial protein according to claim 1 or 2, characterized in that the amino acid sequence of the non-functional amino acid fragment is shown in SEQ ID NO. 6. 4. The artificial protein according to claim 1, wherein the amino acid sequence of the artificial protein is as shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10. 5. Application of the artificial protein described in any one of claims 1 to 4 in the preparation of drugs for restoring immune cell function. 6. The application according to claim 5, characterized in that the immune cells include T cells and/or NK cells. 7. The application according to claim 5 or 6, characterized in that the drug includes drugs that simultaneously identify exhausted immune cells and/or enhance the function of immune cells. 8. Application according to claim 5, characterized in that the medicines include medicines for treating cancer and/or chronic viral infections. 9. The application according to claim 8, wherein the cancer includes one of renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, bladder cancer and lung cancer, or Various. 10. A drug for restoring immune cell function, characterized in that the drug includes the artificial protein according to any one of claims 1 to 4.