Application of POU2F2 protein or mutant thereof in prevention and treatment of infection

Abstract

The invention discloses an application of POU2F2 protein or a mutant thereof in prevention and treatment of infection. The invention provides any one of the following applications of a POU2F2 protein or a mutant thereof, a nucleic acid molecule for coding the POU2F2 protein or the mutant thereof, or an accelerant thereof: (A) an application in preparing a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection; (B) application in preparation of a product for enhancing the anti-infection ability of immune cells; (C) an application in preparation of a product for preventing and / or reversing immune cell depletion.

Description

Technical Field

[0001] The present invention belongs to the technical field of immune cell technology, and particularly relates to the use of POU2F2 protein or its mutants in the prevention and treatment of infections. Background Art

[0002] During pathogen infection, insufficient expansion of immune cells (such as T cells), too rapid regression, inability to form effective immune memory, or entry into a state of exhaustion are important reasons why pathogens cannot be completely cleared and long-term immune memory cannot be formed. Currently, methods for increasing the expansion of immune cells (such as T cells), promoting the formation of immune memory, and inhibiting exhaustion are very limited and further research is needed.

[0003] POU domain, class 2, transcription factor 2 (POU2F2) is a transcription factor that regulates gene expression. Citation 1 discloses a molecular target for preventing and / or treating fibrosis, hypertrophic scar or keloid, comprising: a reagent that inhibits the activity of at least one gene selected from HIC1, FOXS1, CREB5, IRF7, POU2F2, STAT4, TCF4, and / or a reagent that enhances the activity of at least one gene selected from MAF, MEOX2, SIX2. Citation 2 discloses that highly expressed POU2F2 is significantly associated with poor prognosis in patients with glioblastoma multiforme (GBM). POU2F2 promotes cell proliferation and regulates glycolytic reprogramming.

[0004] However, the physiological and pathological functions of POU2F2 in T cells and its role in anti-infection are still unclear.

[0005] Citation

[0006] Citation 1: CN105793279A

[0007] Citation 2: Yang, Rui et al. “POU2F2 regulates glycolytic reprogramming and glioblastoma progression via PDPK1-dependent activation of PI3K / AKT / mTOR pathway.” Cell death & disease vol. 12, 5 433. 30 Apr. 2021, doi: 10.1038 / s41419-021-03719-3 Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] During the infection process, the insufficient expansion of immune cells (such as T cells), the rapid entry into the contraction phase, and the inability to form memory T cells are current problems. The present invention enhances the T cell expansion ability, inhibits T cells from entering the contraction phase, enhances the persistence of T cells, and / or enhances the formation of memory T cells by increasing the level or function of POU2F2 protein during the infection of pathogens (such as viruses and bacteria), thereby enhancing the anti-infection ability of T cells.

[0010] Solutions for Solving the Problems

[0011] In the first aspect of the present invention, there is provided any one of the following uses of POU2F2 protein or its mutant, nucleic acid molecule encoding POU2F2 protein or its mutant, or its promoter:

[0012] (A) Use in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection;

[0013] (B) Use in the preparation of a product for enhancing the anti-infection ability of immune cells;

[0014] (C) Use in the preparation of a product for preventing and / or reversing immune cell exhaustion.

[0015] In some embodiments, the POU2F2 protein is selected from:

[0016] (a) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 12; or

[0017] (b) A protein or polypeptide that is homologous to or has sequence identity (such as more than 80% homology or more than 80% sequence identity, such as 80%, 85%, 90%, 95%, 98%, 99%) with the amino acid sequence shown in SEQ ID NO: 2 or 12, and has at least one activity of anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion; or

[0018] (c) A protein or polypeptide derived from (a) or (b) by substituting, deleting, or adding one or several amino acids in the amino acid sequence of (a) or (b), and having at least one activity of anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion.

[0019] In some embodiments, the nucleic acid molecule is selected from:

[0020] (i) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 9 or 11; or

[0021] (ii) A molecule that hybridizes with the nucleotide sequence defined in (i) under stringent conditions;

[0022] (iii) A nucleic acid molecule that is homologous to or has sequence identity with the nucleotide sequence shown in SEQ ID NO: 9 or 11 (for example, having more than 80% homology or more than 80% sequence identity, such as 80%, 85%, 90%, 95%, 98%, 99%), and encodes a protein or polypeptide having at least one activity among anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion;

[0023] (iv) A nucleic acid molecule obtained by substituting, deleting, or adding one or several nucleotides in the nucleotide sequence of (i) or (ii) or (iii), and encoding a protein or polypeptide having at least one activity among anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion.

[0024] In some embodiments, the promoter is selected from: a substance that increases the level of POU2F2 protein or promotes the function of POU2F2 protein; exogenous POU2F2 protein; naked DNA of the POU2F2 protein coding sequence; liposome-encapsulated DNA of the POU2F2 protein coding sequence; a POU2F2 protein precursor protein or conjugate or complex that can be converted into POU2F2 protein in vivo.

[0025] In some embodiments, the POU2F2 protein mutants are selected from:

[0026] (1) A mutant obtained by double-mutating the 184th and 185th amino acids of POU2F2 shown in SEQ ID NO: 2 to alanine;

[0027] (2) A mutant obtained by deleting amino acids 281 to 340 of POU2F2 shown in SEQ ID NO: 2;

[0028] (3) A mutant obtained by double-mutating the 335th and 339th amino acids of POU2F2 shown in SEQ ID NO: 2 to alanine.

[0029] In some specific embodiments, the amino acid sequence of the POU2F2 protein mutant is as shown in SEQ ID NO: 3, 5, or 7.

[0030] In some embodiments, the infection includes viral infection, bacterial infection, fungal infection, protozoan infection, parasitic infection, or a combination thereof.

[0031] In some embodiments, the diseases and / or symptoms related to infection are one or more selected from the following group:

[0032] Pathological damage caused by infection; immune cell exhaustion after infection, including reduced proliferative ability, weakened killing ability, and decreased cytokine secretion of immune cells; endotoxin shock or death; inflammatory damage to organs; multiple organ failure, acute and / or chronic inflammatory diseases caused by infection.

[0033] In some embodiments, the immune cells include at least one of T cells, NKT cells, NK cells, innate lymphoid cells (ILCs), and chimeric antigen receptor NK cells (CAR-NK cells).

[0034] In some specific embodiments, the T cells include naïve T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric antigen receptor T cells (CAR-T cells), T cell receptor T cells (TCR-T cells), and antigen-specific T cells.

[0035] In some alternative embodiments, the T cells include activated T cells.

[0036] In some alternative embodiments, the T cells include antigen-specific T cells, preferably antigen-specific T cells against the pathogen causing the infection.

[0037] In some alternative embodiments, the T cells include CD8 + T cells.

[0038] In some embodiments, the product is a pharmaceutical composition, a kit, a reagent, or a test kit.

[0039] In a second aspect of the present invention, there is provided a recombinant immune cell which has an increased level or function of POU2F2 protein compared to an un-recombinant immune cell.

[0040] In some embodiments, the immune cells include at least one of T cells, NKT cells, NK cells, innate lymphoid cells (ILCs), and chimeric antigen receptor NK cells (CAR-NK cells).

[0041] In some specific embodiments, the T cells include naïve T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric antigen receptor T cells (CAR-T cells), T cell receptor T cells (TCR-T cells), and antigen-specific T cells.

[0042] In some specific embodiments, the T cells comprise activated T cells.

[0043] In some specific embodiments, the T cells comprise antigen-specific T cells. Preferably, the antigen-specific T cells are antigen-specific T cells against the pathogens causing the infection.

[0044] In some specific embodiments, the T cells comprise CD8 + T cells.

[0045] In the third aspect of the present invention, there is provided the use of the recombinant immune cells as described in the second aspect of the present invention in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection.

[0046] In the fourth aspect of the present invention, there is provided a pharmaceutical composition or a kit, which comprises:

[0047] (A) A therapeutically or prophylactically effective amount of the POU2F2 protein or its mutant, a nucleic acid molecule encoding the POU2F2 protein or its mutant, and / or its promoter, and / or the recombinant immune cells as described in the second aspect of the present invention;

[0048] (B) A pharmaceutically or immunologically acceptable carrier or excipient.

[0049] Effects of the Invention

[0050] The present invention reveals that overexpression of POU2F2 and its mutants can significantly enhance the expansion ability and persistence of T cells during infection, and antagonize immune cell exhaustion, thus increasing the anti-infection ability of T cells. Therefore, overexpression of POU2F2 and its mutants can effectively prevent or treat infections and infection-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Overexpression of POU2F2 significantly increased the expansion ability and persistence of CD8 + T cells during chronic viral infection with LCMV clone 13.

[0052] Figure 1In [description], A is a schematic diagram of the experimental design. P14 cells were activated and transduced with retroviruses carrying an overexpressed empty vector with GFP marker (designated as Control in the figure) and retroviruses carrying an overexpressed POU2F2 vector with GFP marker (designated as Pou2f2 in the figure). GFP-positive P14 cells were adoptively transferred into B6 mice respectively. 12 to 24 hours later, B6 mice were infected with LCMV clone 13 virus by tail vein injection. GFP-positive P14 cells in the blood were detected by flow cytometry. Figure 1 B in [description] shows the expression level of POU2F2 in GFP-positive P14 cells. Figure 1 C and D in [description] show the representative graph and kinetic curve of GFP-positive P14 cells in the blood during LCMV clone 13 virus infection (n = 5 mice per group). Figure 1 E in [description] shows the statistical analysis results of virus titers in the sera of control and experimental group mice 30 days after LCMV clone 13 virus infection (n = 4 mice per group). Figure 1 In D and E of [description], the data are presented as mean ± standard error of the mean (mean ± SEM); Figure 1 D in [description] is a two-way ANOVA multiple-comparisons test, Figure 1 E in [description] is a two-tailed unpaired Student’s t test.

[0053] Figure 2 During chronic LCMV clone 13 virus infection, the positive regulation of the expansion ability and persistence of CD8 + T cells by overexpressing POU2F2 is independent of the transcriptional activity of POU2F2.

[0054] P14 cells were activated and transduced with retroviruses carrying an overexpressed empty vector with GFP marker (designated as Control in the figure) and retroviruses carrying an overexpressed POU2F2 or its mutant vector with GFP marker. GFP-positive P14 cells were adoptively transferred into B6 mice respectively. 12 to 24 hours later, B6 mice were infected with LCMV clone 13 virus by tail vein injection. GFP-positive P14 cells in the blood were detected by flow cytometry. Figure 2 A and B in [description] show the representative graph and kinetic curve of GFP-positive P14 cells in the blood during LCMV clone 13 virus infection (n = 5 mice per group). Figure 2For B in it, the data are presented as mean ± standard error of the mean (mean ± SEM), and analyzed by two-way ANOVA multiple-comparisons test.

[0055] Figure 3 Overexpression of POU2F2 significantly increased the expansion ability and persistence of CD8 + T cells during acute LCMV Armstrong viral infection.

[0056] Figure 3 Panel A in it shows the experimental design schematic. P14 cells were activated and transduced with retroviruses expressing an empty vector with GFP marker (designated as Control in the figure) and retroviruses expressing POU2F2 overexpression vector with GFP marker (designated as Pou2f2 in the figure). GFP-positive P14 cells were adoptively transferred into B6 mice respectively. Twelve to twenty-four hours later, B6 mice were infected with LCMV Armstrong virus by tail vein injection. GFP-positive P14 cells in the blood were detected by flow cytometry. Figure 3 Panels B and C in it show representative plots and kinetic curves of GFP-positive P14 cells in the blood during LCMV Armstrong virus infection (n = 5 mice per group). Figure 3 For C in it, the data are presented as mean ± standard error of the mean (mean ± SEM), and analyzed by two-way ANOVA multiple-comparisons test.

[0057] Figure 4 Overexpression of POU2F2 significantly increased the expansion ability and persistence of CD8 + T cells during acute LM-OVA bacterial infection.

[0058] Figure 4 Panel A in it shows the experimental design schematic. OT-1 cells were activated and transduced with retroviruses expressing an empty vector with GFP marker (Control) and retroviruses expressing POU2F2 overexpression vector with GFP marker (designated as Pou2f2 in the figure). GFP-positive OT-1 cells were adoptively transferred into B6 mice respectively. Twelve to twenty-four hours later, B6 mice were infected with LM-OVA bacteria by tail vein injection. GFP-positive OT-1 cells in the blood were detected by flow cytometry. Figure 4 Panels B and C in it show representative plots and kinetic curves of GFP-positive OT-1 cells in the blood during LM-OVA bacterial infection (n = 5 mice per group). Figure 4For C in it, the data are expressed as mean ± standard error of the mean (mean ± SEM), and a two-way ANOVA multiple-comparisons test was performed. Detailed implementation manners

[0059] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" used herein means "serving as an example, embodiment, or illustration". Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0060] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can be implemented without some specific details. In other instances, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0061] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the systematic errors inevitable in industrial production.

[0062] In this specification, the meaning expressed by using "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0063] In this specification, the "some specific / preferred implementation manners", "other specific / preferred implementation manners", "implementation manners", etc. mentioned refer to the specific elements (for example, features, structures, properties, and / or characteristics) related to the implementation manner described are included in at least one of the implementation manners described herein, and may exist in other implementation manners or may not exist in other implementation manners. In addition, it should be understood that the elements can be combined in various implementation manners in any suitable way.

[0064] In this specification, the numerical range expressed by using "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0065] In this specification, when the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0066] In this specification, the term "immune cell" refers to any cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; myeloid cells such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes.

[0067] As used herein, the term "lymphocyte" refers to all populations of immature, mature, undifferentiated, and differentiated white blood cells, including tissue-specific and specialized types. By way of non-limiting example, the lymphocytes encompass B cells, T cells, NKT cells, and NK cells.

[0068] As used herein, "administering", "giving", and "treating", when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "giving", and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treating a cell includes contacting the cell with a reagent and contacting the cell with a fluid that contacts the cell. "Administering", "giving", and "treating" also mean treating a cell in vitro and ex vivo by a reagent, diagnostic agent, binding composition, or by another cell. "Treating", when applied to a human, veterinary, or research subject, refers to therapeutic, prophylactic, or preventive measures, research, and diagnostic applications.

[0069] As used herein, "treatment" means administering to a patient an internal or external therapeutic agent, such as a recombinant immune cell of the present invention, to a patient having one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to alleviate one or more disease symptoms, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age, and weight, as well as the ability of the drug to produce the desired effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare provider to evaluate the severity or progression of the symptom.

[0070] As used herein, the term "prevention" refers to prophylactic treatment of a subject who does not currently have and has not had a disease but is at risk of developing a disease or who has had a disease in the past, does not currently have the disease but is at risk of disease recurrence.

[0071] As used herein, "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or condition of a medical disorder. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the disorder to be treated, the overall health of the patient, the method of administration, route, and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0072] In this specification, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disorder. The term "therapeutically effective amount" can include amounts that improve overall therapy; reduce or avoid symptoms, signs, or causes of a disorder; and / or enhance the therapeutic efficacy of another therapeutic agent.

[0073] In this specification, a "prophylactically effective amount" is an amount sufficient to prevent a disorder or one or more symptoms associated with the disorder or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disorder. The term "prophylactically effective amount" can include amounts that improve overall prophylaxis or enhance the prophylactic efficacy of another prophylactic agent.

[0074] In this specification, the term "pharmaceutically acceptable" (or "pharmacologically acceptable", "medicinally acceptable") refers to molecular entities and compositions that, when administered to an animal or a human, do not produce adverse reactions, allergic reactions, or other untoward reactions, as appropriate. The term "pharmaceutically acceptable carrier" as used herein encompasses any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents, and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as a medium for pharmaceutically acceptable substances.

[0075] In this specification, a "subject" or "host" refers to a human or non-human animal, including mammals. For example, primates (such as humans, monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, etc. A "subject" or "host" includes both therapeutic and non-therapeutic types. A "subject" or "host" includes experimental animal models or animals used for the production of biomolecules that express therapeutic diseases, that is, "non-therapeutic hosts" or "non-therapeutic subjects".

[0076] Detailed description of the technical solution of the present invention:

[0077] One of the main objects of the present invention is to provide the use of POU2F2 protein or its mutants, nucleic acid molecules encoding POU2F2 protein or its mutants, or its promoters in anti-infection, and further provide their use in the treatment or prevention of infectious diseases and related diseases or symptoms. The drugs, pharmaceutical compositions or reagents, and kits of the present invention can be used to effectively resist infections and control the occurrence of infectious diseases.

[0078] In some aspects of the present invention, there is provided the use of POU2F2 protein or its mutants, nucleic acid molecules encoding POU2F2 protein or its mutants, or its promoters in preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells.

[0079] In some embodiments, the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter is used for preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells to improve the anti-infection ability of a subject and prevent and / or treat infections.

[0080] In some aspects of the present invention, there is provided the use of the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection.

[0081] In some aspects of the present invention, there is provided the use of the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter in the preparation of a product for preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells.

[0082] In some aspects of the present invention, there is further provided a method for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection, the method comprising administering to a subject in need a prophylactically and / or therapeutically effective amount of the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter.

[0083] In some aspects of the present invention, there is further provided a method for preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells, the method comprising having an increased level or function of the POU2F2 protein in immune cells, for example, by treating immune cells with a promoter of the POU2F2 protein or its mutant or a promoter of the nucleic acid molecule encoding the POU2F2 protein or its mutant.

[0084] In some aspects of the present invention, there is further provided the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter, which is used for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection.

[0085] In some aspects of the present invention, there is further provided the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter, which is used for preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells.

[0086] POU2F2 Protein (Polypeptide)

[0087] In this specification, the terms "POU2F2 protein (polypeptide)" and "POU2F2" are used interchangeably and refer to POU domain, class 2, transcription factor 2, with Gene ID: 5452 and Uniprot reference number: P09086. The POU2F2 protein of the present invention can be a protein encoded by SEQ ID NO: 11 (human full-length cDNA sequence) or SEQ ID NO: 9 (mouse CDS sequence), or a homologous sequence (e.g., a homologous sequence of POU2F2 can be obtained through databases or alignment software known in the art), mutant, or modified form of these proteins that has anti-infection effects. For example, the POU2F2 protein can be selected from: (a) the amino acid sequence shown in SEQ ID NO: 2 or 12; or (b) a protein or polypeptide derived from (a) that has undergone substitution, deletion, or addition of one or several amino acids in the amino acid sequence defined in (a) and has the activity of anti-infection, preventing and / or reversing immune cell exhaustion, and / or enhancing the anti-infection ability of immune cells.

[0088] The protein or polypeptide of the present invention can be a naturally purified product, a chemically synthesized product, or a product produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animals, insects, and mammalian cells). In the present invention, the POU2F2 protein or polypeptide is preferably encoded by the human or mouse Pou2f2 gene or its homologous gene or family gene.

[0089] In some embodiments, exemplary information on the Pou2f2 gene can be seen in Table 1 below.

[0090] Table 1 Pou2f2 gene information

[0091]

[0092] In the present invention, specifically, the human Pou2f2 gene (Gene ID: 5452, updated on November 23, 2023, https: / / www.ncbi.nlm.nih.gov / gene / 5432) and the mouse Pou2f2 gene (Gene ID: 18987, updated on November 23, 2023, https: / / www.ncbi.nlm.nih.gov / gene / 18987) encode POU2F2 in cells. The above genes are hereby incorporated into the present invention by reference in their entirety.

[0093] Variant forms (e.g., mutants) of the protein or polypeptide of the present invention include, but are not limited to: deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or close properties, the function of the protein or polypeptide usually remains unchanged. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein or polypeptide. For example, the POU2F2 protein or polypeptide of the present invention may or may not include the starting methionine residue and still have the activity of anti-infection, preventing and / or reversing immune cell exhaustion and / or enhancing the anti-infection ability of immune cells.

[0094] Random mutagenesis can be generated by radiation or exposure to mutagens, and the protein or polypeptide in (b) above can also be obtained by site-directed mutagenesis or other known molecular biology techniques. Exemplarily, a transgenic animal can be constructed using the coding sequence encoding the protein or polypeptide, and the transgenic animal can be observed for its resistance to pathogen infection or whether its resistance to pathogen infection is improved to screen and identify the obtained protein or polypeptide.

[0095] Depending on the host used in the recombinant production protocol, the protein or polypeptide of the present invention can be glycosylated or non-glycosylated. The term also includes active fragments and active derivatives of the POU2F2 protein.

[0096] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences that can hybridize with the POU2F2 protein coding sequence under high or low stringency conditions, and polypeptides or proteins obtained using anti-serum against the POU2F2 protein. The present invention can also use other polypeptides, such as fusion proteins containing the POU2F2 protein or its fragments. In addition to almost full-length polypeptides, the present invention also includes soluble fragments of the POU2F2 protein. Generally, the fragment has at least about 10 consecutive amino acids of the POU2F2 protein sequence, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.

[0097] In some specific embodiments, mutants of the POU2F2 protein are selected from:

[0098] (1) The mutant obtained by mutating the 184th and 185th amino acids of POU2F2 to alanine (i.e., POU2F2-184A185A), and the amino acid sequence of POU2F2-184A185A is shown in SEQ ID NO:3;

[0099] (2) The mutant obtained by deleting the amino acids at positions 281 to 340 of POU2F2 (i.e., POU2F2△281-340aa), and the amino acid sequence of POU2F2△281-340aa is shown in SEQ ID NO:5;

[0100] (3) The mutant obtained by mutating the 335th and 339th amino acids of POU2F2 to alanine (i.e., POU2F2-335A 339A), and the amino acid sequence of POU2F2-335A339A is shown in SEQ ID NO:7.

[0101] Nucleic Acid Molecule Encoding POU2F2 Protein or Its Mutant

[0102] In this specification, the terms "POU2F2 gene", "POU2F2 coding gene", "POU2F2 protein coding gene" or "nucleic acid molecule encoding POU2F2" can be used interchangeably, and all refer to a nucleotide sequence encoding the POU2F2 protein or polypeptide (or POU2F2 protein mutant) described in the present invention, which can be, for example, the nucleotide sequence shown in SEQ ID NO:11 human CDS sequence, SEQ ID NO:9 (mouse CDS) sequence, a molecule hybridizing with these sequences under stringent conditions, or a family gene molecule highly homologous to the above molecules. The expression of the said gene has a certain promoting effect on anti-infection, preventing and / or reversing immune cell exhaustion and / or enhancing the anti-infection ability of immune cells. The POU2F2 gene is highly conserved in humans and mice. The human Gene ID is 5452, and the mouse Gene ID is 18987.

[0103] The POU2F2 gene of the present invention can be selected from: (i) SEQ ID NO:9 or SEQ ID NO:11; or (ii) a molecule that hybridizes with the sequence defined in (i) under stringent conditions and has the activities of anti-infection, preventing and / or reversing immune cell exhaustion and / or enhancing the anti-infection ability of immune cells.

[0104] As used herein, the term "stringent conditions" refers to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, and more preferably more than 95%.

[0105] The full-length nucleotide sequence of the POU2F2 gene of the present invention or its fragment can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequence. When the sequence is relatively long, it is often necessary to perform PCR amplification twice or more times, and then splice the fragments amplified each time together in the correct order.

[0106] It should be understood that the POU2F2 gene of the present invention is preferably obtained from humans or mice, and other genes highly homologous to the human or mouse POU2F2 gene (such as having more than 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, and more preferably more than 85% such as 85%, 90%, 95%, 98% or even 99% or more sequence identity) obtained from other animals are also within the equivalent scope preferably considered in the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

[0107] Promoter of POU2F2 Protein or Its Mutant or Coding Sequence of POU2F2 Protein or Its Mutant

[0108] The present invention also relates to "promoters" of the POU2F2 protein or its mutants or the coding sequences of the POU2F2 protein or its mutants. The terms "promoter" or "promoter of the POU2F2 protein or its mutants or the coding sequence of the POU2F2 protein or its mutants" are used interchangeably and refer to substances that can increase the level or activity of the POU2F2 protein or its mutants or the nucleic acid molecules encoding the POU2F2 protein or its mutants. Promoters that can be used in the present invention include but are not limited to: expression vectors of the POU2F2 protein or its mutants, exogenous POU2F2 protein or its mutants, naked DNA of the POU2F2 protein or its mutants or the coding sequence of the POU2F2 protein or its mutants, liposome-encapsulated DNA of the POU2F2 protein or its mutants or the coding sequence of the POU2F2 protein or its mutants, and the POU2F2 protein or its mutants.

[0109] The POU2F2 protein or its mutant, or the promoter of the POU2F2 protein or its mutant, of the present invention can resist infection, and thus can be further used for preventing or treating diseases related to pathogen infection, and / or related symptoms caused by pathogen infection, as well as acute and chronic inflammatory diseases caused by infection, and / or their symptoms.

[0110] Infection

[0111] In this specification, the term "infection" refers to the invasion of a subject's cells, tissues, and / or organs by pathogens such as bacteria, viruses, fungi, worms, or protozoa. In some embodiments, the pathogen can grow, reproduce, and / or produce toxins in the subject's cells, tissues, and / or organs. In some embodiments, the subject can react to the pathogen (i.e., allergic reaction or immune response). Examples of infections include but are not limited to bacterial infection, viral infection, fungal infection, parasitic infection, and protozoal infection.

[0112] In some embodiments, the infection is an acute infection. In other embodiments, the infection is a chronic infection.

[0113] In some embodiments, the infection is a viral infection, and in other embodiments, the infection is a bacterial infection.

[0114] In some embodiments, the diseases and / or symptoms related to infection are one or more selected from the following group: pathological damage caused by infection; immune cell exhaustion after infection, including reduced proliferation ability, weakened killing ability, and reduced cytokine secretion of immune cells; endotoxin shock or death; inflammatory damage of organs; multiple organ failure, for example, the organs are selected from: liver, spleen, brain, kidney, heart, lung, stomach, intestine; acute and / or chronic inflammatory diseases caused by infection (such as autoimmune diseases such as inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, tuberculosis, chronic gastrointestinal diseases).

[0115] In some embodiments, the POU2F2 protein or its mutant, the nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter can enhance the anti-infection ability of a subject, including enhancing the anti-infection ability of immune cells in the subject, and / or preventing and / or reversing immune cell exhaustion in the subject.

[0116] Immune Cells

[0117] In the present invention, the immune cells include T cells, NKT cells, NK cells, innate lymphoid cells (ILCs) or other immune cells with anti-infection ability, and chimeric antigen receptor NK cells (CAR-NK cells) or other therapeutic immune cells expressing non-natural antigen receptors, but are not limited thereto.

[0118] In the present invention, the term "T cell" includes naive T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric antigen receptor T cells (CAR-T cells), T cell receptor T cells (TCR-T cells) and antigen-specific T cells, but are not limited thereto.

[0119] In some specific embodiments, the T cells include activated T cells.

[0120] In some specific embodiments, the T cells include antigen-specific T cells.

[0121] In some specific embodiments, the antigen-specific T cells are antigen-specific T cells against the pathogen causing the infection.

[0122] In some embodiments, the T cells are from a subject. In some specific embodiments, the subject is a human or non-human animal.

[0123] In some specific embodiments, the subject is infected with a pathogen.

[0124] In some specific embodiments, the T cells include antigen-specific T cells against the pathogen causing the infection in a subject infected with the pathogen.

[0125] In some specific embodiments, the T cells include CD8 + T cells.

[0126] In some embodiments, the T cells include exhausted T cells.

[0127] In the present invention, immune cell depletion refers to a pathological state in which the number of certain immune cells in the body's immune system is significantly reduced or their function is impaired, resulting in a decline in the body's immune function.

[0128] In the present invention, the term "exhausted T cell" or "T cell exhaustion" refers to dysfunctional T cells, and exhausted T cells gradually lose their effector function during chronic infection.

[0129] In some specific embodiments, preventing and / or reversing immune cell exhaustion includes: inhibiting T cells from entering the contraction phase, maintaining T cells in the expansion phase, enhancing the T cell expansion ability, enhancing the persistence of T cells, and / or enhancing the formation of memory T cells.

[0130] In the present invention, the "expansion phase" refers to the stage in which T cells undergo an immune response after contacting an antigen or other stimulating factors. In this stage, the number of T cells rapidly increases to counter the antigenic stimulus. This stage is one of the key links in the T cell immune response.

[0131] In the present invention, the "contraction phase" refers to a stage in the T cell immune response, in which the number of T cells significantly decreases. This stage usually occurs after the T cell expansion phase and is a key stage in T cell differentiation.

[0132] In the present invention, "persistence" refers to the lifespan and stability of T cells. T cells with strong persistence can survive for a long time and maintain a certain immune function, which helps to maintain long-term immune memory. T cells with weak persistence are prone to death, and new T cells need to be continuously replenished to maintain the function of the immune system.

[0133] Product

[0134] In the present invention, the product can be a pharmaceutical composition, a kit, a reagent, or a test kit.

[0135] <Recombinant immune cells and their use in anti-infection>

[0136] In some aspects of the present invention, a recombinant immune cell is provided, which has an increased level or function of POU2F2 protein compared to an un-recombinant immune cell.

[0137] In some embodiments, the un-recombinant immune cell can be a naturally occurring, unmodified immune cell in a subject. In some embodiments, the un-recombinant immune cell can be an immune cell that has not been treated with a promoter of the POU2F2 protein or its mutant, or a nucleic acid molecule encoding the POU2F2 protein or its mutant according to the present invention.

[0138] In some specific embodiments, the recombinant immune cell can have enhanced activity of the POU2F2 protein or its mutant and / or an enhanced expression level of the coding sequence of the POU2F2 protein or its mutant.

[0139] In some aspects of the present invention, there is provided the use of recombinant immune cells in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms associated with infection, wherein the recombinant immune cells have an increased level or function of POU2F2 protein.

[0140] In some embodiments, the POU2F2 protein or its mutant, and the coding sequence of the POU2F2 protein or its mutant are as described above.

[0141] In the present invention, the immune cells include T cells, NKT cells, NK cells, innate lymphoid cells (ILCs) or other immune cells with anti-infection ability, and chimeric antigen receptor NK cells (CAR-NK cells) or other therapeutic immune cells expressing non-natural antigen receptors, but are not limited thereto.

[0142] In the present invention, the term "T cell" includes naive T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric antigen receptor T cells (CAR-T cells), T cell receptor T cells (TCR-T cells) and antigen-specific T cells, but are not limited thereto.

[0143] In some embodiments, the T cells include activated T cells.

[0144] In some embodiments, the T cells include CD8 + T cells.

[0145] In some embodiments, the T cells include antigen-specific T cells.

[0146] In some embodiments, the antigen-specific T cells are antigen-specific T cells against the pathogen causing the infection. Wherein, the infection is the infection as described above.

[0147] In some embodiments, the T cells include exhausted T cells.

[0148] In some embodiments, the T cells are from a subject. In some specific embodiments, the subject is a human or a non-human animal.

[0149] In some specific embodiments, the subject has an infection. In some specific embodiments, the T cells include antigen-specific T cells against the pathogen causing the infection in a subject having an infection.

[0150] In some embodiments, the recombinant immune cells can be immune cells treated with a promoter of the POU2F2 protein or its mutant, or a promoter of a nucleic acid molecule encoding the POU2F2 protein or its mutant, wherein the treated immune cells have an increased level or function of the POU2F2 protein (e.g., having enhanced activity of the POU2F2 protein or its mutant and / or enhanced expression level of the coding sequence of the POU2F2 protein or its mutant).

[0151] In some specific embodiments, compared with immune cells not treated with the promoter (non-recombinant immune cells), the expression or function of the POU2F2 protein or its mutant in immune cells treated with the promoter (recombinant immune cells) is increased by at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%.

[0152] (Treatment method)

[0153] In the present invention, the method for treating immune cells with the promoter is not particularly limited. For example, the promoter can be introduced into immune cells. In some exemplary embodiments, treating immune cells with the promoter can be to introduce a nucleotide carrying one or more components capable of expressing the promoter into immune cells by techniques known to those skilled in the art.

[0154] In some embodiments, the vector used is a viral vector, viroid vector or non-viral vector. In some embodiments, the recombinant vector containing a polynucleotide encoding one or more components of the promoter is a viral vector. Suitable viral vectors include, but are not limited to, viral vectors based on the following: vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus and mammary tumor virus), etc. Suitable non-viral vectors are selected from transposons, lipid nanoparticles, liposomes, exosomes, attenuated bacteria or virus-like particles.

[0155] In some embodiments, the polynucleotide sequence encoding the promoter is operably linked to a control element, such as a transcriptional control element, such as a promoter. The transcriptional control element can be functional in eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaeal cells). In some embodiments, the polynucleotide sequence encoding the promoter is operably linked to multiple control elements, which allow the polynucleotide to be expressed in both prokaryotic and eukaryotic cells. Depending on the cell type and gene regulatory system used, any of a number of suitable transcriptional and translational control elements (including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc.) can be used in the expression vector.

[0156] In some embodiments, non-limiting examples of suitable eukaryotic promoters (promoters that function in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, and mouse metallothionein 1. The selection of a suitable vector and promoter is entirely within the capabilities of one of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcriptional terminator. The expression vector may also include appropriate sequences for amplification of the expression. The expression vector may also contain a nucleotide sequence encoding a protein tag (e.g., 6×His tag, hemagglutinin tag, green fluorescent protein, etc.) fused to the site-specific modified polypeptide, thereby producing a chimeric polypeptide.

[0157] (Methods and Cultures for Obtaining Immune Cells).

[0158] In the present invention, there is no particular limitation in principle for the method of obtaining immune cells. Exemplarily, peripheral blood mononuclear cells can be isolated from a subject's peripheral blood, and immune cells of a specific phenotype can be isolated by, for example, magnetic bead sorting, flow cytometry sorting techniques.

[0159] In some embodiments, in the present disclosure, there is no particular limitation in principle for the method of culturing immune cells. In some embodiments, immune cells can be implanted into a subject for expansion, and the expanded recombinant immune cells in vivo can be obtained. The recombinant immune cells obtained after expansion from the first-generation subject can be used for autologous treatment of the subject or for allogeneic treatment of other subjects. In some embodiments, the immune cells are autologous immune cells or allogeneic immune cells for the subject. In some embodiments, the immune cells can also be expanded in vitro.

[0160] Vector, Host and Transgenic Animal

[0161] The present invention also relates to a vector containing the gene encoding the POU2F2 protein or its mutant, a host cell genetically engineered with the vector, and a transgenic animal obtained by transgenesis and highly expressing the POU2F2 protein or its mutant.

[0162] By conventional recombinant DNA techniques, the coding sequences of the present invention can be used to express or produce recombinant POU2F2 protein or its mutant. Generally, the following steps are involved:

[0163] (1) transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the POU2F2 protein or its mutant of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0164] (2) culturing the host cell in a suitable medium;

[0165] (3) isolating and purifying the protein or polypeptide from the medium or the cells.

[0166] In the present invention, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, phages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is usually that it contains an origin of replication, a promoter, a marker gene and translation control elements.

[0167] Methods well known to those skilled in the art can be used to construct an expression vector containing the coding sequence of the POU2F2 protein or its mutant and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In the present invention, the pMIGW vector expression system is preferably used.

[0168] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0169] Vectors containing the appropriate DNA sequences described above, along with appropriate promoters or control sequences, can be used to transform appropriate host cells to enable them to express proteins or polypeptides. The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc. In the present invention, Escherichia coli bacterial cells and human liver cells are preferably used as host cells.

[0170] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs in length, which act on promoters to enhance gene transcription. Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0171] In the present invention, the terms "transgenic animal" or "transformed animal" are used interchangeably, and both refer to cells, organs, tissues, or individuals that have been obtained by conventional transgenic methods and into which the POU2F2 gene of the present invention has been introduced and which stably and highly express the POU2F2 protein or polypeptide.

[0172] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography, and various other liquid chromatography techniques and combinations of these methods.

[0173] <Drug composition or kit>

[0174] The present invention also provides a product, which can be, for example, a reagent, a drug, a drug composition, or a kit, which contains an effective amount of the POU2F2 protein or its mutant of the present invention, or a nucleic acid molecule encoding the POU2F2 protein or its mutant, or its promoter, or the recombinant immune cells of the present invention, and a pharmaceutically or immunologically acceptable carrier. As used herein, the terms "active substance" or "active substance of the present invention" are used interchangeably and refer to the POU2F2 protein or its mutant, or the coding sequence of the POU2F2 protein or its mutant, or its promoter, or the recombinant immune cells.

[0175] In a preferred embodiment, the product can be used for preventing or treating diseases related to infection, acute and chronic inflammatory diseases caused by infection, and / or their symptoms; for example, the pharmaceutical composition of the present invention can be used for preventing or treating infectious diseases known in the prior art that can be treated or prevented, such as tissue damage caused by infection; inflammatory damage of organs; multiple organ failure.

[0176] In some embodiments, the product is a pharmaceutical composition or a kit, such as a pharmaceutical composition or a kit in a form suitable for administration by a method selected from the group consisting of: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by replication-defective bacteria, DNA of interest carried by replication-defective adenovirus or protein encoded by the gene of interest, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, intratumoral administration.

[0177] The active substance in the product of the present invention accounts for 0.001 to 99.9 wt% of the total weight of the composition; preferably 1 to 95 wt% of the total weight of the composition, more preferably 5 to 90 wt%, and even more preferably 10 to 80 wt%. The balance is pharmaceutically acceptable carriers and other additives and the like.

[0178] As used herein, the term "unit dosage form" refers to a dosage form prepared from the product of the present invention for convenient administration in a single dose, including but not limited to various solid forms (such as tablets), liquid forms, capsules, sustained-release forms.

[0179] It should be understood that the effective dose of the active substance such as the POU2F2 protein or its mutant or the POU2F2 protein or its mutant coding sequence, recombinant immune cells, etc. used can vary depending on the severity of the subject to be administered or treated. The specific situation is determined according to the individual situation of the subject (such as the subject's body weight, age, physical condition, the effect to be achieved), which is within the scope that can be judged by a skilled physician.

[0180] The products of the present invention can be in solid state (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid state (such as oral liquid) or other suitable forms. The administration routes can be: (1) direct naked DNA or protein injection method; (2) connecting the cDNA, mRNA and protein of POU2F2 with transferrin / poly-L-lysine complex to enhance its biological effect; (3) forming a complex of cDNA, mRNA and protein with positively charged lipids to overcome the difficulty of crossing cell membranes caused by the negative charge of the phosphate backbone; (4) mediating the entry of cDNA, mRNA and protein into cells after encapsulating them with liposomes, which is beneficial for the smooth entry of macromolecules and protects them from hydrolysis by various extracellular enzymes; (5) binding cDNA, mRNA and protein with cholesterol to increase the cytoplasmic retention time by 10 times; (6) transporting cDNA, mRNA and protein with immunoliposomes can specifically transport them to target tissues and target cells; (7) in vitro transfection of cDNA, mRNA and protein into transfected cells (such as fibroblasts) can also load POU2F2-related drugs into target cells better; (8) electroporation, that is, introducing cDNA, mRNA and protein into target cells by means of electric current; (9) cellular immunotherapy, injecting recombinant immune cells into the human body.

[0181] Example

[0182] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0183] Example 1. Preparation of recombinant T cells overexpressing POU2F2 and its mutants

[0184] 1. Construction of gene overexpression vectors

[0185] In this example, overexpression vectors based on retroviruses were constructed, namely pMIGW-GFP, pMIGW-POU2F2-GFP, pMIGW-POU2F2-184A 185A-GFP, pMIGW-POU2F2△281-340aa-GFP and pMIGW-POU2F2-335A339A-GFP.

[0186] Among them, the vector pMIGW-GFP (Addgene, plasmid #12282) is an empty vector sequence that does not encode an overexpressed gene.

[0187] The vector pMIGW-POU2F2-GFP (SEQ ID NO:1), positions 6622-8013 are the full-length coding sequence of POU2F2.

[0188] The amino acid sequence of POU2F2 (SEQ ID NO:2):

[0189] MVHSSMGAPEIRMSKPLEAEKQSLDSPSEHTDTERNGPDINHQNPQNKASPFSVSPTGPSTKIKAEDPSGDSAPAAPPPPQPAQPHLPQAQLMLTGSQLAGDIQQLLQLQQLVLVPGHHLQPPAQFLLPQAQQSQPGLLPTPNLFQLPQQTQGALLTSQPRAGLPTQPPKCLEPPSHPEEPSDLEELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDAETMSVDSSLPSPNQLSSPSLGFDGLPGRRRKKRTSIETNVRFALEKSFLANQKPTSEEILLIAEQLHMEKEVIRVWFCNRRQKEKRINPCSAAPMLPSPGKPTSYSPHLVTPQGGAGTLPLSQASSSLSTTVTTLSSAVGTLHPSRTAGGGGGGGGAAPPLNSIPSVTPPPPATTNSTNPSPQGSHSAIGLSGLNPSAGPGLWWNPAPYQP

[0190] The vector pMIGW-POU2F2-184A 185A-GFP, positions 6622-8013 of SEQ ID NO:1 are replaced with the coding sequence (SEQ ID NO:4) of the double mutation of the 184th and 185th amino acids of POU2F2 to alanine (i.e., POU2F2-184A 185A).

[0191] The amino acid sequence of POU2F2-184A 185A (SEQ ID NO:3): MVHSSMGAPEIRMSKPLEAEKQSLDSPSEHTDTERNGPDINHQNPQNKASPFSVSPTGPSTKIKAEDPSGDSAPAAPPPPQPAQPHLPQAQLMLTGSQLAGDIQQLLQLQQLVLVPGHHLQPPAQFLLPQAQQSQPGLLPTPNLFQLPQQTQGALLTSQPRAGLPTQPPKCLEPPSHPEEPSD AAELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDAETMSVDSSLPSPNQLSSPSLGFDGLPGRRRKKRTSIETNVRFALEKSFLANQKPTSEEILLIAEQLHMEKEVIRVWFCNRRQKEKRINPCSAAPMLPSPGKPTSYSPHLVTPQGGAGTLPLSQASSSLSTTVTTLSSAVGTLHPSRTAGGGGGGGGAAPPLNSIPSVTPPPPATTNSTNPSPQGSHSAIGLSGLNPSAGPGLWWNPAPYQP

[0192] The underlined positions are the mutation sites.

[0193] The nucleotide sequence encoding POU2F2-184A 185A (SEQ ID NO:4): ATGGTTCATTCCAGCATGGGGGCTCCAGAAATAAGAATGTCTAAGCCCCTGGAGGCCGAGAAGCAAAGTCTGGACTCCCCGTCAGAGCACACAGACACCGAAAGAAATGGACCCGACATTAACCATCAGAACCCCCAGAATAAAGCGTCCCCATTCTCTGTGTCCCCAACTGGCCCCAGCACCAAGATCAAGGCTGAAGACCCCAGTGGCGATTCAGCCCCAGCAGCACCCCCGCCCCCCCAGCCGGCTCAGCCTCATCTGCCCCAGGCCCAACTCATGCTGACGGGCAGCCAGCTAGCTGGGGACATACAGCAACTCCTCCAGCTCCAGCAGCTGGTGCTTGTCCCCGGCCACCACCTCCAGCCACCTGCTCAGTTCCTGCTGCCACAGGCACAGCAGAGTCAGCCAGGCCTGCTACCAACGCCAAATCTATTCCAGCTACCTCAACAAACCCAGGGAGCTCTCCTGACCTCCCAGCCCCGGGCTGGGCTTCCTACACAGCCCCCGAAATGCTTGGAGCCGCCCTCCCACCCGGAGGAGCCCAGCGAT GCAGCAGAGCTGGAACAGTTTGCTCGCACCTTCAAGCAACGCCGCATCAAGCTGGGCTTCACACAGGGTGATGTGGGCCTGGCCATGGGCAAGCTCTATGGCAACGACTTCAGCCAAACGACCATTTCCCGCTTCGAGGCCCTCAACCTGAGCTTCAAGAACATGTGTAAACTCAAGCCCCTCCTGGAGAAGTGGCTCAACGACGCAGAGACTATGTCTGTGGATTCAAGCCTACCCAGCCCAAACCAGCTGAGCAGCCCCAGCCTGGGTTTCGACGGGCTGCCGGGGCGGAGACGCAAGAAGAGGACCAGCATCGAGACGAATGTCCGCTTCGCCTTAGAGAAGAGTTTCCTAGCGAACCAGAAGCCTACCTCAGAGGAGATCCTGCTGATCGCAGAGCAGCTGCACATGGAGAAGGAAGTGATCCGCGTCTGGTTCTGCAACCGGCGCCAGAAGGAGAAACGCATCAACCCTTGCAGTGCGGCCCCCATGCTGCCCAGCCCGGGAAAGCCGACCAGCTACAGCCCTCACCTGGTCACACCCCAAGGGGGCGCAGGGACCTTACCATTGTCCCAAGCTTCTAGCAGTCTGAGCACAACAGTTACTACCTTATCCTCAGCTGTGGGGACGCTCCATCCCAGCCGGACAGCAGGAGGGGGTGGGGGTGGGGGCGGAGCTGCGCCCCCCCTCAATTCCATCCCCTCTGTCACTCCCCCACCCCCGGCCACCACCAACAGCACAAACCCGAGCCCTCAAGGCAGCCACTCGGCTATTGGCTTGTCGGGCCTGAACCCCAGCGCGGGCCCTGGCCTCTGGTGGAACCCTGCCCCTTACCAGCCTTGA

[0194] Among them, the underlined sites are the mutation sites.

[0195] Vector pMIGW-POU2F2△281-340aa-GFP, the 6622-8013 positions of SEQ ID NO:1 are replaced with the coding sequence (SEQ ID NO:6) of POU2F2 lacking amino acids 281 to 340 (i.e., POU2F2△281-340aa).

[0196] Amino acid sequence of POU2F2△281-340aa (SEQ ID NO:5):

[0197] MVHSSMGAPEIRMSKPLEAEKQSLDSPSEHTDTERNGPDINHQNPQNKASPFSVSPTGPSTKIKAEDPSGDSAPAAPPPPQPAQPHLPQAQLMLTGSQLAGDIQQLLQLQQLVLVPGHHLQPPAQFLLPQAQQSQPGLLPTPNLFQLPQQTQGALLTSQPRAGLPTQPPKCLEPPSHPEEPSDLEELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDAETMSVDSSLPSPNQLSSPSLGFDGLPGRPCSAAPMLPSPGKPTSYSPHLVTPQGGAGTLPLSQASSSLSTTVTTLSSAVGTLHPSRTAGGGGGGGGAAPPLNSIPSVTPPPPATTNSTNPSPQGSHSAIGLSGLNPSAGPGLWWNPAPYQP

[0198] Nucleotide sequence encoding POU2F2△281-340aa (SEQ ID NO:6):

[0199]

[0200] The vector pMIGW-POU2F2-335A 339A-GFP, the coding sequence (SEQ ID NO:8) in which the 6622-8013 positions of SEQ ID NO:1 are replaced with the 335th and 339th amino acid double mutations of POU2F2 to alanine (i.e., POU2F2-335A 339A).

[0201] The amino acid sequence of POU2F2-335A 339A (SEQ ID NO:7):

[0202] MVHSSMGAPEIRMSKPLEAEKQSLDSPSEHTDTERNGPDINHQNPQNKASPFSVSPTGPSTKIKAEDPSGDSAPAAPPPPQPAQPHLPQAQLMLTGSQLAGDIQQLLQLQQLVLVPGHHLQPPAQFLLPQAQQSQPGLLPTPNLFQLPQQTQGALLTSQPRAGLPTQPPKCLEPPSHPEEPSDLEELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDAETMSVDSSLPSPNQLSSPSLGFDGLPGRRRKKRTSIETNVRFALEKSFLANQKPTSEEILLIAEQLHMEKEVIRVWFCNRRQ A EKR A NPCSAAPMLPSPGKPTSYSPHLVTPQGGAGTLPLSQASSSLSTTVTTLSSAVGTLHPSRTAGGGGGGGGAAPPLNSIPSVTPPPPATTNSTNPSPQGSHSAIGLSGLNPSAGPGLWWNPAPYQP

[0203] The underlined positions are the mutation sites.

[0204] The nucleotide sequence encoding POU2F2-335A 339A (SEQ ID NO:8):

[0205] GCA GAGAAACGC GCA AACCCTTGCAGTGCGGCCCCCATGCTGCCCAGCCCGGGAAAGCCGACCAGCTACAGCCCTCACCTGGTCACACCCCAAGGGGGCGCAGGGACCTTACCATTGTCCCAAGCTTCTAGCAGTCTGAGCACAACAGTTACTACCTTATCCTCAGCTGTGGGGACGCTCCATCCCAGCCGGACAGCAGGAGGGGGTGGGGGTGGGGGCGGAGCTGCGCCCCCCCTCAATTCCATCCCCTCTGTCACTCCCCCACCCCCGGCCACCACCAACAGCACAAACCCGAGCCCTCAAGGCAGCCACTCGGCTATTGGCTTGTCGGGCCTGAACCCCAGCGCGGGCCCTGGCCTCTGGTGGAACCCTGCCCCTTACCAGCCTTGA

[0206] The underlined part is the mutation site.

[0207] 2. Isolation and activation of naïve P14 cells

[0208] Naïve CD8 + T cells were isolated from the spleen of Cas9 + P14 transgenic mice (obtained by crossing Jaxson Laboratory, JAX:#037394 and #026430), and the cells were resuspended in 2 ml of RPMI1640 medium (containing 5% fetal bovine serum and interleukin-2). Meanwhile, the polypeptide gp33-41 (Qiangyao Biotech, Cat#04010023714; a polypeptide composed of 9 amino acid residues, which is the best sequence in the lymphocyte choriomeningitis virus GP1 epitope, with the sequence KAVYNFATC; SEQ ID NO:13) was added for in vitro activation, that is, the cells were cultured in an incubator at 37 °C with 5% carbon dioxide. After 24 hours of culture, virus infection was carried out.

[0209] 3. Isolation and activation of naïve OT1 cells

[0210] Naïve CD8 + T cells were isolated from the spleen of Cas9 +T cells were resuspended in 2 ml of RPMI 1640 medium (containing 5% fetal bovine serum and interleukin-2), and at the same time, the polypeptide OVA257-264 (synthesized by Qiangyao Biotech; a polypeptide composed of 8 amino acid residues, which is a class I (Kb)-restricted peptide epitope of ovalbumin (OVA) with the sequence SIINFEKL; SEQ ID NO: 10) was added for in vitro activation. That is, the cells were cultured in an incubator with 5% carbon dioxide at 37 °C. After 24 hours of culture, virus infection was carried out.

[0211] 4. Construction of P14 cells overexpressing POU2F2

[0212] 1) Preparation of retrovirus

[0213] 1×10 6 After Phoenix-Eco cells (ATCC#CRL-3214) were adherently cultured for 24 hours, 20 μg of the expression vector pMIGW-POU2F2-GFP prepared in 1 above and 60 μg of the packaging plasmid pCL-Eco (purchased from Addgene#12371) were co-transfected by the calcium phosphate precipitation method. After 48 hours of transfection, the supernatant containing the packaged virus was harvested, and the virus supernatant was filtered through a 0.45 μm filter membrane to remove dead cell impurities, obtaining the retrovirus supernatant, that is, the retrovirus overexpressing POU2F2.

[0214] 2) Infection with retrovirus particles

[0215] 1×10 of CD8 6 T cells after 36 hours of in vitro activation culture in step 2 were added to 1 ml of the retrovirus supernatant obtained in step 1), mixed well, and centrifuged at 2000×g horizontally at room temperature for 2 hours. Then it was placed in a carbon dioxide incubator and cultured for 4 hours, and then changed to 2 ml of fresh RPMI 1640 medium (containing 5% fetal bovine serum and 2 ng / ml of interleukin-2) and continued to be cultured (this time was recorded as the time after infection). GFP-positive and CD8 + T cells were sorted by flow cytometry, that is, P14 cells overexpressing POU2F2 (denoted as Pou2f2-P14) were obtained. + T cells, that is, P14 cells overexpressing POU2F2 (denoted as Pou2f2-P14) were obtained.

[0216] 5. Construction of P14 cells overexpressing POU2F2-184A 185A

[0217] The difference from "4. Construction of P14 cells overexpressing POU2F2" is only that: replace "expression vector pMIGW-POU2F2-GFP" with "expression vector pMIGW-POU2F2-184A 185A-GFP", and keep other steps unchanged, to obtain P14 cells overexpressing POU2F2-184A 185A (denoted as Pou2f2-184A 185A-P14).

[0218] 6. Construction of P14 cells overexpressing POU2F2△281-340aa

[0219] The difference from "4. Construction of P14 cells overexpressing POU2F2" is only that: replace "expression vector pMIGW-POU2F2-GFP" with "expression vector pMIGW-POU2F2△281-340aa-GFP", and keep other steps unchanged, to obtain P14 cells overexpressing POU2F2△281-340aa (denoted as Pou2f2△281-340aa-P14).

[0220] 7. Construction of P14 cells overexpressing POU2F2-335A 339A

[0221] The difference from "4. Construction of P14 cells overexpressing POU2F2" is only that: replace "expression vector pMIGW-POU2F2-GFP" with "expression vector pMIGW-POU2F2-335A 339A-GFP", and keep other steps unchanged, to obtain P14 cells overexpressing POU2F2-335A 339A (denoted as Pou2f2-335A 339A-P14).

[0222] 8. Construction of control Control-P14 cells

[0223] The difference from "3. Construction of P14 cells overexpressing POU2F2" is only that: replace "expression vector pMIGW-POU2F2-GFP" with "empty vector pMIGW-GFP that does not encode the overexpressed gene", and keep other steps unchanged, to obtain control P14 cells (denoted as Control-P14).

[0224] 9. Construction of OT1 cells overexpressing POU2F2

[0225] The difference from "4. Construction of P14 cells overexpressing POU2F2" is that: replace "P14 cells" with "OT1 cells", and keep other steps unchanged, to obtain P14 cells overexpressing POU2F2 (denoted as Pou2f2-OT1).

[0226] 10. Construction of Control-OT1 Cells

[0227] It is only different from "9. Construction of Control-P14 Cells" in that: "P14 cells" are replaced by "OT1 cells", and other steps remain unchanged, obtaining Control-OT1 cells (denoted as Control-OT1).

[0228] Example 2. Virus Infection and Titration

[0229] Lymphocytic choriomeningitis virus (LCMV) strains Armstrong and LCMV clone 13 each induce acute and chronic infections. LCMV replicates in BHK21[C13] cells ( CCL10) and is titrated by plaque assay on Vero cells. Mice are infected intravenously with LCMV clone 13 (2×10 6 PFU) or intraperitoneally with LCMV Armstrong (2×10 6 PFU). Mice infected with LCMV are raised in accordance with the institutional biosafety regulations of Tsinghua University. The LCMV viral load in serum / organ / tissue samples is quantified by qPCR assay.

[0230] Example 3. Infection with Listeria Expressing Ovalbumin 257-264 Peptide

[0231] Listeria expressing ovalbumin 257-264 peptide is hereinafter referred to as LM-OVA. LM-OVA replicates in brain heart infusion medium supplemented with erythromycin. Mice are infected intravenously with LM-OVA (1×10 5 PFU). Mice infected with LM-OVA are raised in accordance with the institutional biosafety regulations of Tsinghua University. The LM-OVA bacterial load in serum / organ / tissue samples is quantified by qPCR assay.

[0232] Example 4. Overexpression of POU2F2 Significantly Enhances the Expansion and Persistence of CD8 + T Cells during Chronic Infection with LCMV Clone 13 Virus

[0233] The cell transfer process is as shown in A in Figure 1 : CD8 + T cells are isolated from the spleen and lymph nodes of Cas9 + P14 transgenic mice, activated with the polypeptide gp33-41 for 24 hours to obtain activated CD8 + T cells (the method is the same as 2 in Example 1); then the retroviruses obtained by transfecting pMIGW-GFP and the retroviruses obtained by transfecting pMIGW-POU2F2-GFP are respectively used to infect the activated CD8+ T cells were obtained, and the recombinant cells were named Control-P14 and Pou2f2-P14 cells (the method was the same as steps 4 and 8 in Example 1). The above-mentioned cells obtained after 24 hours of infection were respectively injected into B6 mice via the tail vein. The specific injection method was as follows:

[0234] B6 mice at 6-8 weeks old with a body weight of 20-25 g were divided into 2 groups, namely the control (Control) group (5 mice) and the Pou2f2 group (5 mice). The mice were infected with LCMV clone 13 (the method was the same as in Example 2).

[0235] Control group: The Control-P14 cells prepared by the method in Example 1 were made into a cell suspension with PBS and then back-transfused into each mouse in the Control group via the tail vein;

[0236] Pou2f2 group: The Pou2f2-P14 cells prepared by the method in Example 1 were made into a cell suspension with PBS and then back-transfused into each mouse in the Pou2f2 group via the tail vein;

[0237] At 7 days, 14 days, 21 days, and 28 days after the back-transfusion respectively, anti-CD8 antibody and GFP fluorescence flow cytometry were used to analyze the proportion of the back-transfused Control-P14 cells and Pou2f2-P14 cells in the total CD8 + T cells in the peripheral blood of each mouse, that is, to monitor the proliferation, contraction, and persistence of the input P14 cells in the blood.

[0238] The results were as shown in C in Figure 1 and D in Figure 1 During the chronic infection of LCMV clone 13, on the 7th day after infection, P14 in both the control group and the P14 cell group overexpressing POU2F2 (Pou2f2 group) was amplified in the blood; subsequently, on the 14th and 21st days after infection, the P14 cells in the control (Control) group gradually decreased and entered the contraction phase, while overexpression of POU2F2 inhibited the decrease of P14 cells in the contraction phase; until 28 days after infection, the P14 cells in the Pou2f2 group still remained at the level in the expansion phase. It was shown that overexpression of POU2F2 inhibited T cells from entering the contraction phase and enhanced the persistence of P14 cells and the formation of memory P14 cells.

[0239] On the 30th day after infection, we analyzed the virus titer in the serum of each group of mice. As shown in E in Figure 1 :

[0240] Compared with the control group, overexpression of POU2F2 in P14 cells better controlled the replication of LCMV clone 13 virus, indicating that P14 cells overexpressing POU2F2 had antiviral efficacy in mice chronically infected with LCMV clone 13.

[0241] Example 5: Mutants overexpressing POU2F2 also significantly enhanced the expansion and persistence of CD8 + T cells during chronic infection with LCMV clone 13 virus

[0242] Cell re-infusion process: Isolate CD8 + T cells from the spleen and lymph nodes of Cas9 + P14 transgenic mice, activate them with the polypeptide gp33-41 for 24 hours to obtain activated CD8 + T cells (the method is the same as 2 in Example 1); then the retroviruses obtained by transfecting pMIGW-GFP, the retroviruses obtained by transfecting pMIGW-POU2F2-GFP, the retroviruses obtained by transfecting pMIGW-POU2F2-184A 185A-GFP, the retroviruses obtained by transfecting pMIGW-POU2F2△281-340aa-GFP, and the retroviruses obtained by transfecting pMIGW-POU2F2-335A339A-GFP were respectively used to infect the activated CD8 + T cells to obtain recombinant cells named Control-P14, Pou2f2-P14, Pou2f2-184A 185A-P14, Pou2f2-△281-340aa-P14, and Pou2f2-335A339A-P14 cells (the method is the same as 4-8 in Example 1). Perform flow cytometry analysis on the above cells obtained after 24 hours of infection. As Figure 1 shown in B, compared with the control pMIGW-GFP, T cells transfected with the pMIGW-POU2F2-GFP virus highly expressed POU2F2. Infuse them into B6 mice via the tail vein of the mouse, and the specific input method is as follows:

[0243] Divide 6-8-week-old B6 mice weighing 20-25 g into 2 groups, namely the control (Control) group, Pou2f2 group, Pou2f2-184A 185A group, Pou2f2-△281-340aa group, and Pou2f2-335A 339A group, with 5 mice in each group. Infect the mice with LCMV clone 13 (the method is the same as Example 2).

[0244] Control (Control) group: Prepare a cell suspension of Control-P14 cells prepared by the method of Example 1 with PBS and re-infuse it into each mouse in the Control group via the tail vein;

[0245] Pou2f2 group: The Pou2f2-P14 cells prepared by the method of Example 1 were made into a cell suspension with PBS and back-infused into the tail vein of each mouse in the Pou2f2 group;

[0246] Pou2f2-184A 185A group: The Pou2f2-184A 185A-P14 cells prepared by the method of Example 1 were made into a cell suspension with PBS and back-infused into the tail vein of each mouse in the Pou2f2-184A 185A group;

[0247] Pou2f2-△281-340aa group: The Pou2f2-△281-340aa-P14 cells prepared by the method of Example 1 were made into a cell suspension with PBS and back-infused into the tail vein of each mouse in the Pou2f2-△281-340aa group;

[0248] Pou2f2-335A 339A group: The Pou2f2-335A 339A-P14 cells prepared by the method of Example 1 were made into a cell suspension with PBS and back-infused into the tail vein of each mouse in the Pou2f2-335A 339A group;

[0249] At 7 days, 14 days, 21 days, 28 days and 56 days after the back-infusion respectively, anti-CD8 antibody and GFP fluorescence flow cytometry were used to analyze the proportion of the transfected and expressed empty vector virus Control-P14 cells, Pou2f2-P14 cells, Pou2f2-184A 185A-P14 cells, Pou2f2-△281-340aa-P14 cells and Pou2f2-335A 339A-P14 cells in the peripheral blood of each mouse among the total CD8 + T cells, that is, to monitor the proliferation, contraction and persistence of the input P14 cells in the blood.

[0250] The results are as Figure 2 in A and Figure 2As shown in B of [Figure ID], during the chronic infection of LCMV clone 13, on the 7th day after infection, the amplification levels of control, overexpressed POU2F2, and POU2F2-184A 185A P14 cells in the blood were similar, while the amplification of overexpressed POU2F2-△281-340aa P14 cells and POU2F2-335A 339A P14 cells was enhanced; subsequently, on the 14th and 28th days after infection, the P14 cells in the control (Control) group gradually decreased and entered the contraction phase, while overexpression of POU2F2 or several of its mutants inhibited the decrease of P14 cells in the contraction phase; until 56 days after infection, the P14 cells in the control group retracted to about 1%, while the P14 cells overexpressing POU2F2 or several of its mutants still remained at a relatively high proportion, and the proportion of P14 cells overexpressing POU2F2-335A 339A was the highest. The above results indicate that overexpression of POU2F2 or several mutants inhibits the entry of T cells into the contraction phase and enhances the persistence of P14 cells and the formation of memory P14 cells.

[0251] Example 6. Overexpression of POU2F2 significantly enhanced the amplification and persistence of CD8 + T cells during the acute infection of LCMV Amstrong virus

[0252] The cell re-infusion process is as shown in Figure 3 A of [Figure ID]: Isolate CD8 + T cells from the spleen and lymph nodes of Cas9 + P14 transgenic mice, activate them with the polypeptide gp33-41 for 24 hours to obtain activated CD8 + T cells (the method is the same as 2 in Example 1); then infect the activated CD8 + T cells with the retrovirus obtained by transfecting pMIGW-GFP and the retrovirus obtained by transfecting pMIGW-POU2F2-GFP respectively to obtain recombinant cells named Control-P14 and Pou2f2-P14 cells (the method is the same as 4 and 8 in Example 1). Input the above cells obtained after 24 hours of infection into B6 mice via the tail vein of the mouse, and the input method is as follows:

[0253] Divide 6-8-week-old B6 mice weighing 20-25 g into 2 groups, namely the control (Control) group (5 mice) and the Pou2f2 group (5 mice). Infect the mice with LCMV Amstrong (the method is the same as Example 2).

[0254] Control (Control) group: Prepare a cell suspension of Control-P14 cells prepared by the method of Example 1 with PBS and re-inject it into each mouse in the Control group via the tail vein;

[0255] Pou2f2 group: The Pou2f2-P14 cells prepared by the method of Example 1 were configured into a cell suspension with PBS and retro-injected into the tail veins of each mouse in the Pou2f2 group;

[0256] At 7 days, 14 days, 21 days, and 28 days after retro-injection respectively, anti-CD8 antibody and GFP fluorescence flow cytometry were used to analyze the proportion of retro-injected Control-P14 cells and Pou2f2-P14 cells in the peripheral blood of each mouse among the total CD8 + T cells, that is, to monitor the proliferation, contraction, and persistence of the input P14 cells in the blood.

[0257] The results were as Figure 3 shown in B of Figure 3 and C of

[0258] During the acute infection induced by LCMVA mstrong, at 7 days after infection, P14 in both the control group and the overexpressed POU2F2 P14 cell group (Pou2f2 group) was amplified in the blood, and overexpression of POU2F2 enhanced the amplification of P14; subsequently, at 14 days, 21 days, and 28 days after infection, the P14 cells in the control (Control) group gradually decreased and entered the contraction phase, while overexpression of POU2F2 significantly inhibited the decrease of P14 cells in the contraction phase. It shows that overexpression of POU2F2 inhibits T cells from entering the contraction phase and enhances the persistence of P14 cells.

[0258] Example 7. Overexpression of POU2F2 significantly enhanced the amplification and persistence of CD8 + T cells during the acute infection induced by LM-OVA

[0259] The cell retro-injection process was as Figure 4 shown in A of + CD8 + T cells were isolated from the spleen and lymph nodes of Cas9 + OT1 transgenic mice, activated with the polypeptide OVA257-264 for 24 hours to obtain activated OT1 cells (the method was the same as 3 in Example 1); then the retrovirus obtained by transfecting pMIGW-GFP and the retrovirus obtained by transfecting pMIGW-POU2F2-GFP were respectively used to infect the activated OT1 cells, and the recombinant cells were named Control-OT1 and Pou2f2-OT1 cells (the method was the same as 9-10 in Example 1). The above cells obtained after 24 hours of infection were respectively retro-injected into the tail veins of B6 mice, and the input method was as follows:

[0260] Six- to eight-week-old B6 mice weighing 20-25 g were divided into 2 groups, namely the control (Control) group (5 mice) and the Pou2f2 group (5 mice). The mice were infected with LM-OVA (the method was the same as in Example 2).

[0261] Control group: The Control-OT1 cells prepared by the method of Example 1 were configured into a cell suspension with PBS and retrotransfused into the tail vein of each mouse in the Control group;

[0262] Pou2f2 group: The Pou2f2-OT1 cells prepared by the method of Example 1 were configured into a cell suspension with PBS and retrotransfused into the tail vein of each mouse in the Pou2f2 group;

[0263] At 7 days, 14 days, and 28 days after retrotransfusion respectively, anti-CD8 antibody and GFP fluorescence flow cytometry were used to analyze the proportion of retrotransfused Control-OT1 cells and Pou2f2-OT1 cells in the peripheral blood of each mouse among total CD8 + T cells, that is, to monitor the proliferation, contraction, and persistence of the input OT1 cells in the blood.

[0264] The results are as Figure 4 shown in B of Figure 4 and C of

[0265] During the LM-OVA infection process, at 7 days after infection, OT1 in both the control group and the overexpressed POU2F2 P14 cell group (Pou2f2 group) was amplified in the blood, and overexpression of POU2F2 significantly enhanced the amplification of OT1; subsequently, at 14 days and 28 days after infection, OT1 cells in the control (Control) group gradually decreased and entered the contraction phase, while overexpression of POU2F2 significantly inhibited the decrease of OT1 cells in the contraction phase. It shows that overexpression of POU2F2 inhibits T cells from entering the contraction phase and enhances the persistence of OT1 cells.

[0265] Nucleotide sequence of vector pMIGW-POU2F2-GFP (SEQ ID NO:1):

[0266] tctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc atggtgagcaagggcgaggagc tgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtctggcga gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctgg cccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgact tcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagac ccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggac ggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaaga acggcatcaaggcgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagca gaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaa gaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacg agctgtacaagtaaATGGTTCATTCCAGCATGGGGGCTCCAGAAATAAGAATGTCTAAGCCCCTGGAGGCCGA GAAGCAAAGTCTGGACTCCCCGTCAGAGCACACAGACACCGAAAGAAATGGACCCGACATTAACCATCAGAACCCC CAGAATAAAGCGTCCCCATTCTCTGTGTCCCCAACTGGCCCCAGCACCAAGATCAAGGCTGAAGACCCCAGTGGCG ATTCAGCCCCAGCAGCACCCCCGCCCCCCCAGCCGGCTCAGCCTCATCTGCCCCAGGCCCAACTCATGCTGACGGG CAGCCAGCTAGCTGGGGACATACAGCAACTCCTCCAGCTCCAGCAGCTGGTGCTTGTCCCCGGCCACCACCTCCAG CCACCTGCTCAGTTCCTGCTGCCACAGGCACAGCAGAGTCAGCCAGGCCTGCTACCAACGCCAAATCTATTCCAGC TACCTCAACAAACCCAGGGAGCTCTCCTGACCTCCCAGCCCCGGGCTGGGCTTCCTACACAGCCCCCGAAATGCTT GGAGCCGCCCTCCCACCCGGAGGAGCCCAGCGATCTGGAGGAGCTGGAACAGTTTGCTCGCACCTTCAAGCAACGC CGCATCAAGCTGGGCTTCACACAGGGTGATGTGGGCCTGGCCATGGGCAAGCTCTATGGCAACGACTTCAGCCAAA CGACCATTTCCCGCTTCGAGGCCCTCAACCTGAGCTTCAAGAACATGTGTAAACTCAAGCCCCTCCTGGAGAAGTG GCTCAACGACGCAGAGACTATGTCTGTGGATTCAAGCCTACCCAGCCCAAACCAGCTGAGCAGCCCCAGCCTGGGT TTCGACGGGCTGCCGGGGCGGAGACGCAAGAAGAGGACCAGCATCGAGACGAATGTCCGCTTCGCCTTAGAGAAGA GTTTCCTAGCGAACCAGAAGCCTACCTCAGAGGAGATCCTGCTGATCGCAGAGCAGCTGCACATGGAGAAGGAAGT GATCCGCGTCTGGTTCTGCAACCGGCGCCAGAAGGAGAAACGCATCAACCCTTGCAGTGCGGCCCCCATGCTGCCC AGCCCGGGAAAGCCGACCAGCTACAGCCCTCACCTGGTCACACCCCAAGGGGGCGCAGGGACCTTACCATTGTCCC AAGCTTCTAGCAGTCTGAGCACAACAGTTACTACCTTATCCTCAGCTGTGGGGACGCTCCATCCCAGCCGGACAGC AGGAGGGGGTGGGGGTGGGGGCGGAGCTGCGCCCCCCCTCAATTCCATCCCCTCTGTCACTCCCCCACCCCCGGCC ACCACCAACAGCACAAACCCGAGCCCTCAAGGCAGCCACTCGGCTATTGGCTTGTCGGGCCTGAACCCCAGCGCGG GCCCTGGCCTCTGGTGGAACCCTGCCCCTTACCAGCCTTGA gaattccgcccccccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctga

[0267] Among them, the single-underlined part is EGFP; the double-underlined part is the coding sequence of POU2F2, which is also SEQ ID NO:9.

[0268] Nucleotide sequence of POU2F2 (human) SEQ ID NO:11:

[0269]

[0270] Amino acid sequence of POU2F2 (human) SEQ ID NO:12:

[0271] MVHSSMGAPEIRMSKPLEAEKQGLDSPSEHTDTERNGPDTNHQNPQNKTSPFSVSPTGPSTKIKAEDPSGDSAPAAPLPPQPAQPHLPQAQLMLTGSQLAGDIQQLLQLQQLVLVPGHHLQPPAQFLLPQAQQSQPGLLPTPNLFQLPQQTQGALLTSQPRAGLPTQAVTRPTLPDPHLSHPQPPKCLEPPSHPEEPSDLEELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDAETMSVDSSLPSPNQLSSPSLGFDGLPGRRRKKRTSIETNVRFALEKSFLANQKPTSEEILLIAEQLHMEKEVIRVWFCNRRQKEKRINPCSAAPMLPSPGKPASYSPHMVTPQGGAGTLPLSQASSSLSTTVTTLSSAVGTLHPSRTAGGGGGGGGAAPPLNSIPSVTPPPPATTNSTNPSPQGSHSAIGLSGLNPSTGPGLWWNPAPYQP

[0272] It should be noted that although the technical solutions of the present invention are introduced by specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0273] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, the practical application or the improvement of the technology in the market, or to enable other ordinary skilled in the art in this technical field to understand the disclosed embodiments.

Claims

1. Use of any one of the POU2F2 protein or its mutants, nucleic acid molecules encoding the POU2F2 protein or its mutants, or its promoters: (A) Use in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection; (B) Use in the preparation of a product for enhancing the anti-infection ability of immune cells; (C) Use in the preparation of a product for preventing and / or reversing immune cell exhaustion.

2. The use according to claim 1, characterized in that, The POU2F2 protein is selected from: (a) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 12; or (b) A protein or polypeptide that is more than 80% homologous to the amino acid sequence shown in SEQ ID NO: 2 or 12 or has a sequence identity of more than 80%, and has at least one activity among anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion; or (c) A protein or polypeptide derived from (a) or (b) in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of (a) or (b), and has at least one activity among anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion; and / or The nucleic acid molecule is selected from: (i) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 9 or 11; or (ii) A molecule that hybridizes with the nucleotide sequence defined in (i) under stringent conditions; (iii) A nucleic acid molecule that is more than 80% homologous to the nucleotide sequence shown in SEQ ID NO: 9 or 11 or has a sequence identity of more than 80%, and encodes a protein or polypeptide having at least one activity among anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion; (iv) A nucleic acid molecule in which one or several nucleotides are substituted, deleted, or added in the nucleotide sequence of (i) or (ii) or (iii), and encodes a protein or polypeptide having at least one activity among anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion; and / or The promoter is selected from: substances that increase the level of the POU2F2 protein or promote the function of the POU2F2 protein; exogenous POU2F2 protein; naked DNA of the POU2F2 protein coding sequence; liposome-encapsulated DNA of the POU2F2 protein coding sequence; a POU2F2 protein precursor protein or conjugate or complex that can be converted into the POU2F2 protein in vivo.

3. The use according to claim 1 or 2, characterized in that, The POU2F2 protein mutants are selected from: (1) A mutant obtained by double mutation of amino acids 184 and 185 of POU2F2 shown in SEQ ID NO: 2 to alanine; (2) A mutant obtained by deleting amino acids 281 to 340 of POU2F2 shown in SEQ ID NO: 2; (3) A mutant obtained by double mutation of amino acids 335 and 339 of POU2F2 shown in SEQ ID NO: 2 to alanine; Optionally, the amino acid sequence of the POU2F2 protein mutant is as shown in SEQ ID NO: 3, 5, or 7.

4. Use according to any one of claims 1 to 3, characterized in that, The infection includes viral infection, bacterial infection, fungal infection, protozoal infection, parasitic infection, or a combination thereof.

5. The use according to any one of claims 1 to 4, characterized in that, The diseases and / or symptoms associated with the infection are one or more selected from the group consisting of: Pathological damage caused by the infection; immune cell exhaustion after infection, including reduced proliferative capacity of immune cells, weakened killing ability, and reduced cytokine secretion; endotoxin shock or death; inflammatory damage to organs; multiple organ failure, acute and / or chronic inflammatory diseases caused by the infection.

6. The use according to any one of claims 1 to 5, characterized in that, The immune cells include at least one of T cells, NKT cells, NK cells, innate lymphoid cells (ILC), and chimeric antigen receptor NK cells (CAR-NK cells); Optionally, the T cells include naïve T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric antigen receptor T cells (CAR-T cells), T cell receptor T cells (TCR-T cells), and antigen-specific T cells, or at least one of them; Optionally, the T cells comprise activated T cells; Optionally, the T cells comprise antigen-specific T cells, preferably, the antigen-specific T cells are antigen-specific T cells against the pathogen causing the infection; Optionally, the T cells comprise CD8 + T cells.

7. Use according to any one of claims 1 to 6, characterized in that The product is a pharmaceutical composition, a kit, a reagent, or a test kit.

8. A recombinant immune cell, which has an increased level or function of POU2F2 protein compared to an un-recombinant immune cell; Optionally, the immune cells include at least one of T cells, NKT cells, NK cells, innate lymphoid cells (ILC), and chimeric antigen receptor NK cells (CAR-NK cells); Optionally, the T cells include naïve T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, exhausted T cells, tolerant T cells, chimeric antigen receptor T cells (CAR-T cells), T cell receptor T cells (TCR-T cells), and antigen-specific T cells, at least one of which; Optionally, the T cells comprise activated T cells; Optionally, the T cells comprise antigen-specific T cells, preferably, the antigen-specific T cells are antigen-specific T cells against the pathogen causing the infection; Optionally, the T cell comprises CD8 + T cell.

9. Use of the recombinant immune cell according to claim 8 in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms associated with the infection.

10. A pharmaceutical composition or a kit, comprising: (A) A therapeutically or prophylactically effective amount of POU2F2 protein or its mutant, a nucleic acid molecule encoding POU2F2 protein or its mutant, and / or its promoter, and / or the recombinant immune cell according to claim 8; (B) A pharmaceutically or immunologically acceptable carrier or excipient.

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