Use of POU2f2 protein or mutant thereof in prevention and treatment of infections

Abstract

Disclosed is a use of a POU2F2 protein or a mutant thereof in prevention and treatment of infections. Provided is any one of the following uses of the POU2F2 protein or the mutant thereof, a nucleic acid molecule encoding the POU2F2 protein or the mutant thereof, or a promoter thereof: (A) a use in preparation of a product for prevention and / or treatment of infectious diseases and / or infection-related diseases and / or symptoms; (B) a use in preparation of a product for enhancing the anti-infective capability of immune cells; and (C) a use in preparation of a product for prevention and / or reversal of immune cell depletion.

Description

Use of POU2F2 protein or its mutant in preventing and treating infection

[0001] Priority and related applications

[0002] The present invention claims priority to Chinese patent application No. 202410029751.X filed on January 9, 2024, entitled “Use of POU2F2 protein or its mutants in preventing and treating infections”. The entire contents of the above application, including the appendix, are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of immune cell technology, and particularly relates to the use of POU2F2 protein or a mutant thereof in preventing and treating infection. Background Art

[0004] During pathogen infection, insufficient expansion of immune cells (such as T cells), rapid extinction, and the inability to form effective immune memory, or exhaustion, are key factors in preventing pathogens from being completely eliminated and forming long-term immune memory. Currently, methods for increasing the expansion of immune cells (such as T cells), promoting immune memory formation, and inhibiting exhaustion are very limited and require further research.

[0005] POU domain, class 2, transcription factor 2 (POU2F2) is a transcription factor that regulates gene expression. Reference 1 discloses a molecular target for preventing and / or treating fibrosis, hypertrophic scars, or keloids, comprising: an agent that inhibits the activity of at least one gene selected from HIC1, FOXS1, CREB5, IRF7, POU2F2, STAT4, and TCF4, and / or an agent that enhances the activity of at least one gene selected from MAF, MEOX2, and SIX2. Reference 2 discloses that high expression of POU2F2 is significantly associated with poor prognosis in patients with glioblastoma (GBM). POU2F2 promotes cell proliferation and regulates glycolytic reprogramming.

[0006] However, the physiological and pathological functions of POU2F2 in T cells and its role in anti-infection remain unclear.

[0007] Citations

[0008] Reference 1: CN105793279A

[0009] 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.12,5 433.30Apr.2021,doi:10.1038 / s41419-021-03719-3 Summary of the Invention

[0010] Problems to be solved by the invention

[0011] The current problem is that immune cells (such as T cells) fail to expand sufficiently during infection, rapidly enter a contraction phase, and fail to form memory T cells. The present invention significantly enhances T cell expansion, inhibits T cell contraction, increases T cell persistence, and / or enhances the formation of memory T cells during infection with pathogens (such as viruses and bacteria), thereby improving T cell resistance to infection.

[0012] Solutions for solving problems

[0013] In the first aspect of the present invention, any of the following uses of a POU2F2 protein or a mutant thereof, a nucleic acid molecule encoding a POU2F2 protein or a mutant thereof, or a promoter thereof is provided:

[0014] (A) use in the preparation of a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms associated with infection;

[0015] (B) use in the preparation of a product for enhancing the anti-infection ability of immune cells;

[0016] (C) Use in the preparation of a product for preventing and / or reversing immune cell exhaustion.

[0017] In some embodiments, the POU2F2 protein is selected from:

[0018] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 12; or

[0019] (b) a protein or polypeptide that is homologous or has sequence identity (e.g., greater than 80% homologous or having sequence identity, such as 80%, 85%, 90%, 95%, 98%, or 99%) to the amino acid sequence of SEQ ID NO: 2 or 12, and has at least one of the following activities: anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion; or

[0020] (c) A protein or polypeptide derived from (a) or (b) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b) and which has at least one activity of anti-infection, enhancing the anti-infection ability of immune cells and preventing and / or reversing immune cell exhaustion.

[0021] In some embodiments, the nucleic acid molecule is selected from the group consisting of:

[0022] (i) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 9 or 11; or

[0023] (ii) a molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (i);

[0024] (iii) a nucleic acid molecule that is homologous or has sequence identity (e.g., greater than 80% homologous or having sequence identity, such as 80%, 85%, 90%, 95%, 98%, or 99%) to the nucleotide sequence of SEQ ID NO: 9 or 11, and encodes a protein or polypeptide having at least one of the following activities: anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion;

[0025] (iv) a nucleic acid molecule comprising a nucleotide sequence of (i) or (ii) or (iii) in which one or more nucleotides are substituted, deleted or added, and encoding a protein or polypeptide having at least one of the activities of anti-infection, enhancing the anti-infection ability of immune cells, and preventing and / or reversing immune cell exhaustion.

[0026] In some embodiments, the promoter is selected from: substances that increase the level of POU2F2 protein or promote 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; POU2F2 protein precursor protein or conjugate or complex that can be converted into POU2F2 protein in vivo.

[0027] In some embodiments, the POU2F2 protein mutant is selected from:

[0028] (1) a mutant obtained by mutating amino acids 184 and 185 of POU2F2 as shown in SEQ ID NO: 2 to alanine;

[0029] (2) a mutant obtained by deleting amino acids 281 to 340 of POU2F2 as shown in SEQ ID NO: 2;

[0030] (3) A mutant obtained by double mutation of amino acids 335 and 339 of POU2F2 to alanine as shown in SEQ ID NO: 2.

[0031] In some specific embodiments, the amino acid sequence of the POU2F2 protein mutant is shown in SEQ ID NO: 3, 5 or 7.

[0032] In some embodiments, the infection comprises a viral infection, a bacterial infection, a fungal infection, a protozoan infection, a parasitic infection, or a combination thereof.

[0033] In some embodiments, the infection-related diseases and / or symptoms are one or more selected from the group consisting of:

[0034] Pathological damage caused by infection; immune cell exhaustion after infection, including reduced proliferation and killing ability of immune cells, and reduced cytokine secretion; endotoxic shock or death; inflammatory damage to organs; multiple organ failure, and acute and / or chronic inflammatory diseases caused by infection.

[0035] In some embodiments, the immune cells include at least one of T cells, NKT cells, NK cells, innate lymphocytes (ILCs), and chimeric antigen receptor NK cells (CAR-NK cells).

[0036] In some specific embodiments, the T cells include naive T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + At least one of 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.

[0037] In some optional embodiments, the T cells comprise activated T cells.

[0038] In some optional embodiments, the T cells comprise antigen-specific T cells. Preferably, the antigen-specific T cells are antigen-specific T cells against pathogens that cause infection.

[0039] In some optional embodiments, the T cells comprise CD8 + T cells.

[0040] In some embodiments, the product is a pharmaceutical composition, a kit, a reagent, or a test kit.

[0041] In a second aspect of the present invention, a recombinant immune cell is provided, wherein the recombinant immune cell has an increased level or function of POU2F2 protein compared to a non-recombinant immune cell.

[0042] In some embodiments, the immune cells include at least one of T cells, NKT cells, NK cells, innate lymphocytes (ILCs), and chimeric antigen receptor NK cells (CAR-NK cells).

[0043] In some specific embodiments, the T cells comprise naive T cells, CD4 + T cells, CD8 + At least one of 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.

[0044] In some specific embodiments, said T cells comprise activated T cells.

[0045] In some specific embodiments, the T cells comprise antigen-specific T cells. Preferably, the antigen-specific T cells are antigen-specific T cells against pathogens that cause infection.

[0046] In some specific embodiments, the T cells comprise CD8 + T cells.

[0047] In a third aspect, the present invention provides use of the recombinant immune cell as described in the second aspect of the present invention in preparing a product for preventing and / or treating infectious diseases and / or diseases and / or symptoms associated with infection.

[0048] In a fourth aspect of the present invention, a pharmaceutical composition or kit is provided, comprising:

[0049] (A) a therapeutically or prophylactically effective amount of a POU2F2 protein or a mutant thereof, a nucleic acid molecule encoding a POU2F2 protein or a mutant thereof and / or a promoter thereof, and / or a recombinant immune cell as described in the second aspect of the present invention;

[0050] (B) a pharmaceutically or immunologically acceptable carrier or excipient.

[0051] Effects of the Invention

[0052] The present invention reveals that overexpression of POU2F2 and its mutants can significantly enhance the proliferation and persistence of T cells during infection and antagonize immune cell exhaustion, thereby increasing T cell resistance to infection. Therefore, overexpression of POU2F2 and its mutants can effectively prevent or treat infections and infection-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1. Overexpression of POU2F2 significantly increased CD8 + T cell expansion capacity and persistence.

[0054] Figure 1A is a schematic diagram of the experimental design. P14 cells were activated and transduced with a retrovirus containing an empty vector overexpressing the GFP marker (designated "Control") and a retrovirus containing a vector overexpressing the GFP marker POU2F2 (designated "Pou2f2"). GFP-positive P14 cells were adoptively transferred into B6 mice. 12 to 24 hours later, the B6 mice were infected with LCMV clone 13 virus via tail vein injection. GFP-positive P14 cells in the blood were detected by flow cytometry. Figure 1B shows the expression of POU2F2 in GFP-positive P14 cells. Figures 1C and D show representative images and kinetic curves of GFP-positive P14 cells in the blood during LCMV clone 13 infection (n = 5 mice per group). Figure 1E shows the statistical analysis of viral titers in the serum of control and experimental mice 30 days after LCMV clone 13 infection (n = 4 mice per group). In Figure 1D and E, the data represent mean ± SEM; Figure 1D is a two-way ANOVA multiple-comparisons test, and Figure 1E is a two-tailed unpaired Student's t test.

[0055] Figure 2. Overexpression of POU2F2 affects CD8 + Positive regulation of T cell expansion and persistence is independent of POU2F2 transcriptional activity.

[0056] P14 cells were activated and transduced with a retrovirus containing an empty vector overexpressing a GFP marker (represented as Control in the figure) and a retrovirus containing a vector overexpressing POU2F2 or its mutants with a GFP marker. GFP-positive P14 cells were adoptively transferred into B6 mice, and 12 to 24 hours later, the 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. Figures 2A and 2B show representative images and kinetic curves of GFP-positive P14 cells in the blood during LCMV clone 13 virus infection (n=5 mice per group). In Figure 2B, the data represent the mean ± standard error (mean ± SEM), and the two-way ANOVA multiple-comparisons test was used.

[0057] Figure 3. Overexpression of POU2F2 significantly increased CD8 expression during acute LCMV Armstrong infection. + T cell expansion capacity and persistence.

[0058] Figure 3 A is a schematic diagram of the experimental design. P14 cells were activated and transduced with a retrovirus containing an empty vector overexpressing a GFP marker (represented as Control in the figure) and a retrovirus containing a POU2F2 overexpression vector overexpressing a GFP marker (represented as Pou2f2 in the figure). GFP-positive P14 cells were adoptively transferred into B6 mice, and 12 to 24 hours later, the B6 mice were infected with LCMV Armstrong virus by tail vein injection. GFP-positive P14 cells in the blood were detected by flow cytometry. Figures 3 B and C show representative images and kinetic curves of GFP-positive P14 cells in the blood during LCMV Armstrong virus infection (n = 5 mice per group). In Figure 3 C, the data represent the mean ± standard error (mean ± SEM), and the two-way ANOVA multiple-comparisons test was used.

[0059] Figure 4. Overexpression of POU2F2 significantly increased CD8 + T cell expansion capacity and persistence.

[0060] Figure 4A is a schematic diagram of the experimental design. OT-1 cells were activated and transduced with a retrovirus containing an empty vector overexpressing the GFP marker (Control) or a retrovirus containing a vector overexpressing the GFP marker POU2F2 (denoted as Pou2f2 in the figure). GFP-positive OT-1 cells were adoptively transferred into B6 mice. 12 to 24 hours later, the mice were infected with LM-OVA bacteria via tail vein injection. GFP-positive OT-1 cells in the blood were detected by flow cytometry. Figures 4B and C show representative images and kinetic curves of GFP-positive OT-1 cells in the blood during LM-OVA infection (n = 5 mice per group). In Figure 4C, data are presented as mean ± standard error (SEM) using a two-way ANOVA multiple-comparisons test. DETAILED DESCRIPTION

[0061] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0062] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain 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 main points of the present invention.

[0063] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0064] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0065] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0066] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0067] 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.

[0068] As used herein, the term "immune cell" refers to any cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes.

[0069] In this specification, the term "lymphocyte" refers to all immature, mature, undifferentiated and differentiated white lymphocyte populations, including tissue-specific and specialized types. By way of non-limiting example, the lymphocytes encompass B cells, T cells, NKT cells and NK cells.

[0070] As used herein, "administer," "give," and "treat" as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by a reagent, a diagnostic agent, a binding composition, or by another cell. "Treat" as applied to a human, veterinary, or research subject refers to treatment, prophylaxis, or preventative measures, research, and diagnostic applications.

[0071] As used herein, "treatment" means administering an internal or external therapeutic agent, such as a recombinant immune cell comprising the present invention, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the patient or population being treated, 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 according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms.

[0072] In this specification, the term "prevention" refers to preventive treatment of a subject who does not currently have a disease or has not had a disease in the past but is at risk of developing a disease or who has had a disease in the past and does not currently have a disease but is at risk of recurrence of the disease.

[0073] As used herein, an "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0074] As used herein, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with the condition. 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 condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and / or enhances the therapeutic efficacy of another therapeutic agent.

[0075] As used herein, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition 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 condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0076] In this specification, the term "pharmaceutically acceptable" (or "pharmacologically acceptable", "pharmaceutically usable") refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals or humans, as appropriate. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0077] As used herein, "subject" or "host" refers to a human or non-human animal, including mammals. Examples include primates (e.g., humans and monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, and mice. "Subjects" or "hosts" include both therapeutic and non-therapeutic subjects. "Subjects" or "hosts" also include experimental animal models or animals used to produce biomolecules expressing therapeutic diseases, i.e., "non-therapeutic hosts" or "non-therapeutic subjects."

[0078] The technical solution of the present invention is described in detail:

[0079] One of the primary objectives of the present invention is to provide anti-infective uses of the POU2F2 protein or its mutants, nucleic acid molecules encoding the POU2F2 protein or its mutants, or promoters thereof, and further provide their use in treating or preventing infectious diseases and related diseases or symptoms. The drugs, pharmaceutical compositions, reagents, and kits of the present invention can be used to effectively combat infection and control the occurrence of infectious diseases.

[0080] 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 promoters thereof in preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells.

[0081] In some embodiments, POU2F2 protein or its mutant, nucleic acid molecules encoding POU2F2 protein or its mutant, or promoters thereof are used to prevent and / or reverse immune cell exhaustion or enhance the anti-infection ability of immune cells to improve the subject's anti-infection ability, prevent and / or treat infection.

[0082] In some aspects of the present invention, provided are uses of POU2F2 protein or its mutants, nucleic acid molecules encoding POU2F2 protein or its mutants, or promoters thereof in the preparation of products for preventing and / or treating infectious diseases and / or diseases and / or symptoms associated with infection.

[0083] In some aspects of the present invention, the use of POU2F2 protein or its mutants, nucleic acid molecules encoding POU2F2 protein or its mutants, or promoters thereof in the preparation of products for preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells is provided.

[0084] In some aspects of the present invention, a method for preventing and / or treating infectious diseases and / or infection-related diseases and / or symptoms is also provided, which comprises administering to a subject in need thereof an effective amount of POU2F2 protein or its mutant, a nucleic acid molecule encoding POU2F2 protein or its mutant, or a promoter thereof.

[0085] In some aspects of the present invention, a method for preventing and / or reversing immune cell exhaustion or enhancing the anti-infection ability of immune cells is also provided, which method comprises increasing the level or function of POU2F2 protein in the immune cells, for example, by treating the immune cells with a promoter of POU2F2 protein or its mutant or a promoter of a nucleic acid molecule encoding POU2F2 protein or its mutant.

[0086] In some aspects of the present invention, POU2F2 protein or its mutant, nucleic acid molecules encoding POU2F2 protein or its mutant, or promoters thereof are also provided, which are used to prevent and / or treat infectious diseases and / or diseases and / or symptoms associated with infection.

[0087] In some aspects of the present invention, POU2F2 protein or its mutant, nucleic acid molecules encoding POU2F2 protein or its mutant or promoters thereof are also provided, which are used to prevent and / or reverse immune cell exhaustion or enhance the anti-infection ability of immune cells.

[0088] POU2F2 protein (peptide)

[0089] In this specification, the terms "POU2F2 protein (polypeptide)" and "POU2F2" are used interchangeably and refer to POU domain, class 2, transcription factor 2, Gene ID: 5452, Uniprot reference number: P09086. The POU2F2 protein of the present invention can be a protein encoded by SEQ ID NO: 11 (full-length human cDNA sequence) or SEQ ID NO: 9 (mouse CDS sequence) or a homologous sequence of these proteins having anti-infective effects (for example, homologous sequences of POU2F2 can be obtained through databases or alignment software known in the art), mutants or modified forms. 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 one or more amino acids substituted, deleted or added 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-infective ability of immune cells.

[0090] The protein or polypeptide of the present invention can be a naturally purified product, a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animal, insect, and mammalian cells). The POU2F2 protein or polypeptide of the present invention is preferably encoded by the human or mouse Pou2f2 gene or its homologous gene or family gene.

[0091] In some embodiments, exemplary information of the Pou2f2 gene can be found in Table 1 below.

[0092] Table 1 Pou2f2 gene information

[0093] 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 all incorporated into the present invention by reference.

[0094] Variants (e.g., mutants) of the proteins or polypeptides of the present invention include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), as well as additions of one or more amino acids (generally within 20, preferably within 10, and more preferably within 5) to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter the function of the protein or polypeptide. For example, additions of one or more amino acids to the C-terminus and / or N-terminus generally do not alter the function of the protein or polypeptide. For example, the POU2F2 protein or polypeptide of the present invention may or may not include an initial methionine residue and still exhibit anti-infection, prevent and / or reverse immune cell exhaustion, and / or enhance the anti-infective capacity of immune cells.

[0095] Random mutagenesis can be induced by irradiation or exposure to a mutagen, or the protein or polypeptide described in (b) above can be obtained by site-directed mutagenesis or other known molecular biology techniques. For example, a coding sequence encoding the protein or polypeptide can be used to construct a transgenic animal, and the resulting protein or polypeptide can be screened and identified by observing whether the transgenic animal exhibits resistance to pathogen infection or whether its resistance to pathogen infection is improved.

[0096] Depending on the host used in the recombinant production protocol, the protein or polypeptide of the present invention may be glycosylated, or may be non-glycosylated. The term also includes active fragments and active derivatives of the POU2F2 protein.

[0097] Variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences that hybridize to the POU2F2 protein coding sequence under high or low stringency conditions, and polypeptides or proteins obtained using antisera raised against the POU2F2 protein. Other polypeptides can also be used in the present invention, such as fusion proteins comprising the POU2F2 protein or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses soluble fragments of the POU2F2 protein. Typically, the fragment comprises at least about 10 contiguous amino acids of the POU2F2 protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0098] In some specific embodiments, the mutant of POU2F2 protein is selected from:

[0099] (1) A mutant obtained by mutating amino acids 184 and 185 of POU2F2 to alanine (i.e., POU2F2-184A 185A). The amino acid sequence of POU2F2-184A 185A is shown in SEQ ID NO: 3.

[0100] (2) A mutant obtained by deleting amino acids 281 to 340 of POU2F2 (i.e., POU2F2Δ281-340aa), the amino acid sequence of POU2F2Δ281-340aa is shown in SEQ ID NO: 5;

[0101] (3) A mutant obtained by mutating amino acids 335 and 339 of POU2F2 to alanine (ie, POU2F2-335A 339A). The amino acid sequence of POU2F2-335A 339A is shown in SEQ ID NO: 7.

[0102] Nucleic acid molecules encoding POU2F2 protein or its mutants

[0103] In this specification, the terms "POU2F2 gene," "POU2F2 encoding gene," "POU2F2 protein encoding gene," or "POU2F2 encoding nucleic acid molecule" are used interchangeably and refer to a nucleotide sequence encoding the POU2F2 protein or polypeptide (or POU2F2 protein mutant) of the present invention, which may be, for example, the human CDS sequence of SEQ ID NO: 11, the nucleotide sequence shown in SEQ ID NO: 9 (mouse CDS) sequence, a molecule that hybridizes with these sequences under stringent conditions, or a family gene molecule that is highly homologous to the above-mentioned molecules. The expression of the gene has a certain promoting effect on resisting 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.

[0104] 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 anti-infection activity, prevents and / or reverses immune cell exhaustion and / or enhances the anti-infection ability of immune cells.

[0105] As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent 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 two sequences is at least 50%, preferably greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85% or greater than 90%, and more preferably greater than 95%.

[0106] The full-length nucleotide sequence of the POU2F2 gene or fragments thereof of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. For long sequences, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0107] It should be understood that the POU2F2 gene of the present invention is preferably obtained from humans or mice. Other genes obtained from other animals that are highly homologous to the human or mouse POU2F2 gene (e.g., having a sequence identity of 50% or more, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, and more preferably 85% or more, such as 85%, 90%, 95%, 98%, or even 99% or more) are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0108] Promoter of POU2F2 protein or its mutant or POU2F2 protein or its mutant coding sequence

[0109] The present invention also relates to a "promoter" of the POU2F2 protein or its mutants or the POU2F2 protein or its mutant POU2F2 coding sequence. The terms "promoter" or "promoter of the POU2F2 protein or its mutants or the POU2F2 protein or its mutant coding sequence" are used interchangeably and refer to a substance that can increase the level or activity of the POU2F2 protein or its mutants or the nucleic acid molecule encoding the POU2F2 protein or its mutants. The promoters that can be used in the present invention include, but are not limited to: a POU2F2 protein or its mutant expression vector, an exogenous POU2F2 protein or its mutants, naked DNA of the POU2F2 protein or its mutants or the POU2F2 protein or its mutant coding sequence, liposome-encapsulated DNA of the POU2F2 protein or its mutants or the POU2F2 protein or its mutant coding sequence, and a POU2F2 protein or its mutants.

[0110] The POU2F2 protein or its mutant or POU2F2 protein or its mutant or its promoter of the present invention can resist infection and can be further used to prevent or treat 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.

[0111] Infect

[0112] In this specification, the term "infection" refers to the invasion of cells, tissues and / or organs of a subject by pathogens such as bacteria, viruses, fungi, worms or protozoa. In certain embodiments, pathogens can grow, reproduce and / or produce toxins in cells, tissues and / or organs of a subject. In certain embodiments, the subject can react to the pathogen (i.e., an allergic reaction or an immune response). Examples of infection include but are not limited to bacterial infection, viral infection, fungal infection, parasitic infection and protozoan infection.

[0113] In some embodiments, the infection is an acute infection. In other embodiments, the infection is a chronic infection.

[0114] In some embodiments, the infection is a viral infection, and in other embodiments, the infection is a bacterial infection.

[0115] In some embodiments, the infection-related diseases and / or symptoms are one or more selected from the following groups: pathological damage caused by infection; immune cell exhaustion after infection, including reduced proliferation capacity, weakened killing capacity, and reduced secretion of cytokines of immune cells; endotoxic shock or death; inflammatory damage to 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 (for example, autoimmune diseases such as inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis, tuberculosis, chronic gastrointestinal diseases).

[0116] 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 subject's anti-infection ability, including enhancing the anti-infection ability of immune cells in the subject, and / or prevent and / or reverse the exhaustion of immune cells in the subject.

[0117] immune cells

[0118] In the present invention, the immune cells include T cells, NKT cells, NK cells, innate lymphocytes (ILCs) or other immune cells with anti-infection ability, as well as chimeric antigen receptor NK cells (CAR-NK cells) or other therapeutic immune cells expressing non-natural antigen receptors, but are not limited thereto.

[0119] In the present invention, the term "T cells" includes naive T cells, αβT cells, γδT 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.

[0120] In some specific embodiments, the T cells comprise activated T cells.

[0121] In some specific embodiments, the T cells comprise antigen-specific T cells.

[0122] In some specific embodiments, the antigen-specific T cells are antigen-specific T cells directed against a pathogen that causes an infection.

[0123] In some embodiments, the T cells are from a subject. In some specific embodiments, the subject is a human or non-human animal.

[0124] In some embodiments, the subject is infected with a pathogen.

[0125] In some specific embodiments, the T cells comprise T cells specific for an antigen of the pathogen causing the infection in a subject infected with the pathogen.

[0126] In some specific embodiments, the T cells comprise CD8 + T cells.

[0127] In some embodiments, the T cells comprise exhausted T cells.

[0128] 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 functions are impaired, leading to a decline in the body's immune function.

[0129] In the present invention, the term "exhausted T cells" or "T cell exhaustion" refers to dysfunctional T cells, which gradually lose their effector functions during chronic infection.

[0130] 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 T cell expansion capacity, enhancing T cell persistence and / or enhancing the formation of memory T cells.

[0131] In the present invention, the "expansion phase" refers to the stage in which T cells initiate an immune response after exposure to an antigen or other stimulus. During this stage, the number of T cells increases rapidly to counteract the antigenic stimulus. This phase is a key step in the T cell immune response.

[0132] In the present invention, "contraction phase" refers to a stage in the immune response of T cells, during which the number of T cells decreases significantly. This stage usually occurs after the expansion phase of T cells and is a key stage of T cell differentiation.

[0133] In this context, "persistence" refers to the lifespan and stability of T cells. T cells with strong persistence can survive for long periods of time and maintain certain immune functions, which helps maintain long-term immune memory. T cells with weak persistence are prone to death, requiring constant replenishment of new T cells to maintain immune system function.

[0134] product

[0135] In the present invention, the product may be a pharmaceutical composition, a pharmaceutical kit, a reagent or a test kit.

[0136] <Recombinant immune cells and their anti-infection uses>

[0137] In some aspects of the present invention, a recombinant immune cell is provided, wherein the recombinant immune cell has an increased level or function of POU2F2 protein compared to a non-recombinant immune cell.

[0138] In some embodiments, the non-recombinant immune cell can be a naturally occurring, unmodified immune cell in a subject. In some embodiments, the non-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 promoter of a nucleic acid molecule encoding the POU2F2 protein or its mutant, as described herein.

[0139] In some specific embodiments, the recombinant immune cells can have enhanced activity of the POU2F2 protein or its mutant and / or enhanced expression level of the POU2F2 protein or its mutant coding sequence.

[0140] In some aspects of the present invention, provided are uses of recombinant immune cells in the preparation of products for preventing and / or treating infectious diseases and / or diseases and / or symptoms associated with infections, wherein the recombinant immune cells have increased POU2F2 protein levels or functions.

[0141] In some embodiments, the POU2F2 protein or its mutant, or the POU2F2 protein or its mutant encoding sequence is as described above.

[0142] In the present invention, the immune cells include T cells, NKT cells, NK cells, innate lymphocytes (ILCs) or other immune cells with anti-infection ability, as well as chimeric antigen receptor NK cells (CAR-NK cells) or other therapeutic immune cells expressing non-natural antigen receptors, but are not limited thereto.

[0143] In the present invention, the term "T cells" includes naive T cells, αβT cells, γδT 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.

[0144] In some embodiments, the T cells comprise activated T cells.

[0145] In some embodiments, the T cells comprise CD8 + T cells.

[0146] In some embodiments, the T cells comprise antigen-specific T cells.

[0147] In some embodiments, the antigen-specific T cells are antigen-specific T cells directed against a pathogen causing an infection, wherein the infection is as described above.

[0148] In some embodiments, the T cells comprise exhausted T cells.

[0149] In some embodiments, the T cells are from a subject. In some specific embodiments, the subject is a human or non-human animal.

[0150] In some embodiments, the subject has an infection. In some embodiments, the T cells comprise T cells specific for an antigen of a pathogen causing the infection in a subject having an infection.

[0151] In some embodiments, the recombinant immune cells can be immune cells treated with a promoter of POU2F2 protein or its mutant, or a promoter of a nucleic acid molecule encoding POU2F2 protein or its mutant, wherein the treated immune cells have an increased level or function of POU2F2 protein (for example, having enhanced activity of POU2F2 protein or its mutant and / or enhanced expression level of the POU2F2 protein or its mutant coding sequence).

[0152] In some specific embodiments, the expression or function of POU2F2 protein or its mutant in immune cells (recombinant immune cells) treated with the promoter 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% compared to immune cells (non-recombinant immune cells) not treated with the promoter.

[0153] (Handling method)

[0154] In the present invention, the method for treating immune cells with the promoter is not particularly limited, and for example, the promoter may be introduced into immune cells. In some exemplary embodiments, treating immune cells with the promoter may be introducing nucleotides carrying one or more components capable of expressing the promoter into the immune cells using techniques known to those skilled in the art.

[0155] In some embodiments, the vector used is a viral vector, a virus-like vector or a non-viral vector. In some embodiments, the recombinant vector comprising a polynucleotide encoding one or more components of the promoter is a viral vector. Suitable viral vectors include, but are not limited to, those based on the following viral vectors: 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 leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). Suitable non-viral vectors are selected from transposons, lipid nanoparticles, liposomes, exosomes, attenuated bacteria, or virus-like particles.

[0156] In some embodiments, the polynucleotide sequence encoding the promoter is operably linked to a control element, for example, a transcriptional control element, such as a promoter. Transcriptional control elements 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 a plurality of control elements that allow the polynucleotide to be expressed in both prokaryotic and eukaryotic cells. Depending on the cell type and gene regulatory system used, any one of many suitable transcriptional and translational control elements (including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc.) can be used in the expression vector.

[0157] 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 (LTR) from retroviruses, and mouse metallothionein 1. The selection of suitable vectors and promoters is entirely within the capabilities of those of ordinary skill in the art. The expression vector may also include a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., 6×His tag, hemagglutinin tag, green fluorescent protein, etc.) fused to the site-directed modified polypeptide to produce a chimeric polypeptide.

[0158] (Methods for obtaining and culturing immune cells).

[0159] In the present invention, there is no particular limitation on the method for obtaining immune cells. For example, peripheral blood mononuclear cells can be isolated from the peripheral blood of a subject, and immune cells of a specific phenotype can be isolated by, for example, magnetic bead sorting or flow cytometry sorting.

[0160] In some embodiments, in the present disclosure, there are no particular limitations on the method for culturing immune cells. In some embodiments, immune cells can be implanted into a subject for amplification, and recombinant immune cells after in vivo amplification can be obtained. Recombinant immune cells obtained after amplification from a 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 amplified in vitro.

[0161] Vectors, hosts, and transgenic animals

[0162] The present invention also relates to a vector comprising a gene encoding a POU2F2 protein or a mutant thereof, a host cell produced by genetic engineering using the vector, and a transgenic animal highly expressing a POU2F2 protein or a mutant thereof obtained by genetic engineering.

[0163] The coding sequence of the present invention can be used to express or produce recombinant POU2F2 protein or its mutants by conventional recombinant DNA technology. Generally speaking, the following steps are involved:

[0164] (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a POU2F2 protein or a mutant thereof of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0165] (2) host cells cultured in a suitable culture medium;

[0166] (3) Isolate and purify proteins or peptides from culture medium or cells.

[0167] In this invention, the terms "vector" and "recombinant expression vector" are used interchangeably to refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0168] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the POU2F2 protein or its mutants and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked 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 transcriptional terminator. The pMIGW vector expression system is preferably used in the present invention.

[0169] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of 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.

[0170] Vectors containing the appropriate DNA sequences described above and appropriate promoters or control sequences can be used to transform appropriate host cells to express proteins or polypeptides. Host cells can be prokaryotes, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include Escherichia coli, Streptomyces, and Agrobacterium; fungal cells, such as yeast; and animal cells. In the present invention, Escherichia coli bacterial cells and human liver cells are preferred host cells.

[0171] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription can be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA elements, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Those skilled in the art will readily appreciate how to select appropriate vectors, promoters, enhancers, and host cells.

[0172] In the present invention, the terms "transgenic animals" or "transformed animals" are used interchangeably, and both refer to cells, organs, tissues or individuals obtained by conventional transgenic methods that have been transferred with the POU2F2 gene of the present invention and stably and highly express the POU2F2 protein or polypeptide.

[0173] The recombinant polypeptide in the above method can be expressed intracellularly or on the cell membrane or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing 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 a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, 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.

[0174] <Pharmaceutical Composition or Kit>

[0175] The present invention also provides a product, which can be, for example, a reagent, a drug, a pharmaceutical composition, or a kit containing an effective amount of the POU2F2 protein or a mutant thereof of the present invention, or a nucleic acid molecule encoding the POU2F2 protein or a mutant thereof, or a promoter thereof, or a recombinant immune cell 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 to refer to the POU2F2 protein or a mutant thereof, or a sequence encoding the POU2F2 protein or a mutant thereof, or a promoter thereof, or a recombinant immune cell.

[0176] In a preferred embodiment, the product can be used to prevent or treat diseases associated with 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 to prevent or treat infectious diseases known to be treatable or preventable in the prior art, such as tissue damage caused by infection; inflammatory damage to organs; and multiple organ failure.

[0177] In some embodiments, the product is a pharmaceutical composition or a kit, for example, a pharmaceutical composition or a kit in a form suitable for administration by a method selected from the group consisting of oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, and intratumoral administration.

[0178] The active substance in the product of the present invention accounts for 0.001-99.9 wt% of the total weight of the composition, preferably 1-95 wt%, more preferably 5-90 wt%, and even more preferably 10-80 wt%, with the remainder being pharmaceutically acceptable carriers and other additives.

[0179] As used herein, the term "unit dosage form" refers to the preparation of the product of the present invention into a dosage form required for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release dosage forms.

[0180] It should be understood that the effective dosage of the active substance, such as the POU2F2 protein or its mutant, or the coding sequence for the POU2F2 protein or its mutant, or the recombinant immune cells, may vary depending on the severity of the condition being administered or treated. The specific dosage will be determined based on the individual circumstances of the subject (e.g., weight, age, physical condition, and desired effect), which is within the judgment of a skilled physician.

[0181] The product of the present invention can be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral liquid) or other suitable shapes. The administration route can be: (1) direct naked DNA or protein injection; (2) connecting the cDNA, mRNA and protein of POU2F2 with transferrin / poly-L-lysine complex to enhance its biological effect; (3) forming a complex between cDNA, mRNA and protein and positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating cDNA, mRNA and protein with liposomes and mediating their entry into cells, which is conducive to the smooth entry of macromolecules and avoids the hydrolysis of various extracellular enzymes; (5) cDNA, mRNA and protein RNA and protein bind to cholesterol, increasing their cytoplasmic retention time by 10 times; (6) Using immunoliposomes to transport cDNA, mRNA, and proteins allows their specific transport to target tissues and target cells; (7) In vitro transfection of cDNA, mRNA, and proteins into transfer cells (such as fibroblasts) can also effectively load POU2F2-related drugs into target cells; (8) Electroporation, which is the use of electric current to introduce cDNA, mRNA, and proteins into target cells; (9) Cell immunotherapy, which is the introduction of recombinant immune cells into the human body.

[0182] Example

[0183] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0184] Example 1. Preparation of recombinant T cells overexpressing POU2F2 and its mutants

[0185] 1. Construction of gene overexpression vector

[0186] In this example, retrovirus-based overexpression vectors were constructed, namely, pMIGW-GFP, pMIGW-POU2F2-GFP, pMIGW-POU2F2-184A 185A-GFP, pMIGW-POU2F2Δ281-340aa-GFP, and pMIGW-POU2F2-335A 339A-GFP.

[0187] The vector pMIGW-GFP (Addgene, plasmid #12282) is an empty vector sequence that does not encode an overexpressed gene.

[0188] Vector pMIGW-POU2F2-GFP (SEQ ID NO: 1), positions 6622-8013 are the full-length coding sequence of POU2F2.

[0189] Amino acid sequence of POU2F2 (SEQ ID NO: 2):

[0190] Vector pMIGW-POU2F2-184A 185A-GFP, positions 6622-8013 of SEQ ID NO: 1 were replaced with the coding sequence (SEQ ID NO: 4) in which amino acids 184 and 185 of POU2F2 were double mutated to alanine (ie, POU2F2-184A 185A).

[0191] Amino acid sequence of POU2F2-184A 185A (SEQ ID NO: 3):

[0192] The underlined areas are mutation sites.

[0193] Nucleotide sequence encoding POU2F2-184A 185A (SEQ ID NO: 4):

[0194] The mutation sites are underlined.

[0195] Vector pMIGW-POU2F2Δ281-340aa-GFP, positions 6622-8013 of SEQ ID NO: 1 were replaced with the coding sequence of POU2F2 lacking amino acids 281 to 340 (i.e., POU2F2Δ281-340aa) (SEQ ID NO: 6).

[0196] Amino acid sequence of POU2F2Δ281-340aa (SEQ ID NO: 5):

[0197] Nucleotide sequence encoding POU2F2Δ281-340aa (SEQ ID NO: 6):

[0198] Vector pMIGW-POU2F2-335A 339A-GFP, positions 6622-8013 of SEQ ID NO: 1 were replaced with the coding sequence (SEQ ID NO: 8) in which amino acids 335 and 339 of POU2F2 were double mutated to alanine (ie, POU2F2-335A 339A).

[0199] Amino acid sequence of POU2F2-335A 339A (SEQ ID NO: 7):

[0200] The underlined areas are mutation sites.

[0201] Nucleotide sequence encoding POU2F2-335A 339A (SEQ ID NO: 8):

[0202] The underlined areas are mutation sites.

[0203] 2. Isolation and Activation of Initial P14 Cells

[0204] Cas9 was isolated by magnetic bead sorting + Initial CD8 + T cells were resuspended in 2 ml of RPMI1640 medium (containing 5% fetal bovine serum and interleukin-2) and activated in vitro by the addition of the peptide gp33-41 (Qiangyao Biotechnology, Cat#04010023714; a polypeptide consisting of 9 amino acid residues, which is the optimal sequence in the lymphocytic choriomeningitis virus GP1 antigenic determinant, sequence KAVYNFATC; SEQ ID NO: 13). The cells were cultured in a 5% carbon dioxide incubator at 37°C and infected with the virus after 24 hours of culture.

[0205] 3. Isolation and Activation of Initial OT1 Cells

[0206] Cas9 was isolated by magnetic bead sorting + Initial CD8 + T cells were resuspended in 2 ml of RPMI1640 medium (containing 5% fetal bovine serum and interleukin-2) and activated in vitro by the addition of the polypeptide OVA257-264 (strongly biosynthesized; a polypeptide consisting of 8 amino acid residues, which is a class 1 (Kb) restricted peptide epitope of ovalbumin (OVA), sequence SIINFEKL; SEQ ID NO: 10). The cells were cultured in a 5% carbon dioxide incubator at 37°C and infected with the virus after 24 hours of culture.

[0207] 4. Construction of P14 cells overexpressing POU2F2

[0208] 1) Retrovirus preparation

[0209] 1×10 6 After Phoenix-Eco cells (ATCC #CRL-3214) were cultured adherently 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. The supernatant containing the packaged virus was harvested 48 hours after transfection, and the viral supernatant was filtered with a 0.45 μm filter membrane to remove dead cell impurities to obtain the retroviral supernatant, i.e., the retrovirus overexpressing POU2F2.

[0210] 2) Retroviral infection

[0211] 1×10 6 CD8 + T cells were added with 1 ml of the retroviral supernatant obtained in step 1) and mixed, and then centrifuged at 2000 × g for 2 hours at room temperature. Then, the cells were cultured in a CO2 incubator for 4 hours, replaced with 2 ml of fresh RPMI1640 medium (containing 5% fetal bovine serum and 2 ng / ml interleukin-2) and continued to be cultured (this time was recorded as the post-infection time). GFP-positive and CD8 T cells were sorted by flow cytometry. + T cells, namely P14 cells overexpressing POU2F2 (denoted as Pou2f2-P14), were obtained.

[0212] 5. Construction of P14 cells overexpressing POU2F2-184A 185A

[0213] The only difference from "4. Construction of P14 cells overexpressing POU2F2" is that "expression vector pMIGW-POU2F2-GFP" is replaced with "expression vector pMIGW-POU2F2-184A 185A-GFP" and the other steps remain unchanged to obtain P14 cells overexpressing POU2F2-184A 185A (denoted as Pou2f2-184A 185A-P14).

[0214] 6. Construction of P14 cells overexpressing POU2F2△281-340aa

[0215] The only difference from "4. Construction of P14 cells overexpressing POU2F2" is that the "expression vector pMIGW-POU2F2-GFP" is replaced with the "expression vector pMIGW-POU2F2△281-340aa-GFP", and the other steps remain unchanged, and P14 cells overexpressing POU2F2△281-340aa (indicated as Pou2f2△281-340aa-P14) are obtained.

[0216] 7. Construction of P14 cells overexpressing POU2F2-335A 339A

[0217] The only difference from “4. Construction of P14 cells overexpressing POU2F2” is that “expression vector pMIGW-POU2F2-GFP” is replaced with “expression vector pMIGW-POU2F2-335A 339A-GFP”, and the other steps remain unchanged to obtain P14 cells overexpressing POU2F2-335A 339A (denoted as Pou2f2-335A 339A-P14).

[0218] 8. Construction of Control-P14 cells

[0219] The only difference from "3. Construction of P14 cells overexpressing POU2F2" is that the "expression vector pMIGW-POU2F2-GFP" is replaced with "an empty vector pMIGW-GFP that does not encode an overexpressed gene"; the other steps remain unchanged to obtain control P14 cells (denoted as Control-P14).

[0220] 9. Construction of OT1 cells overexpressing POU2F2

[0221] The difference from “4. Construction of P14 cells overexpressing POU2F2” is that “P14 cells” are replaced with “OT1 cells”, and other steps remain unchanged to obtain P14 cells overexpressing POU2F2 (denoted as Pou2f2-OT1).

[0222] 10. Construction of Control-OT1 cells

[0223] The only difference from "9. Construction of Control-P14 cells" is that "P14 cells" are replaced by "OT1 cells", and other steps remain unchanged to obtain control OT1 cells (denoted as Control-OT1).

[0224] Example 2. Virus infection and titration

[0225] Lymphocytic choriomeningitis virus (LCMV) strains Armstrong and LCMV clone 13 each induced acute and chronic infection. LCMV was expressed in BHK21[C13] cells. The cells were propagated and titrated by plaque assay on VERO cells. LCMV clone 13 (2 × 10 6 Mice were intravenously infected with LCMV Armstrong (2×10 6Mice were infected by intraperitoneal injection of 100 PFU (1000 PFU). LCMV-infected mice were maintained according to the institutional biosafety regulations of Tsinghua University. LCMV viral loads in serum, organ, and tissue samples were quantified by qPCR.

[0226] Example 3: Infection with Listeria expressing the ovalbumin 257-264 peptide segment

[0227] Listeria monocytogenes expressing the 257-264 peptide fragment of ovalbumin, hereinafter referred to as LM-OVA. LM-OVA was propagated in brain heart infusion medium supplemented with erythromycin. 5 Mice were intravenously infected with 100 PFU of LM-OVA. Mice infected with LM-OVA were maintained according to the institutional biosafety regulations of Tsinghua University. LM-OVA bacterial loads in serum, organ, and tissue samples were quantified by qPCR.

[0228] Example 4: Overexpression of POU2F2 significantly enhanced CD8 + T cell expansion and persistence

[0229] The cell reinfusion process is shown in Figure 1A: + CD8 cells were isolated from spleen and lymph nodes of P14 transgenic mice + T cells were activated with peptide gp33-41 for 24 hours to obtain activated CD8 + T cells (the method is the same as that of Example 1, step 2); the retrovirus obtained by transfection with pMIGW-GFP and the retrovirus obtained by transfection with pMIGW-POU2F2-GFP were respectively infected with activated CD8 + The recombinant cells were named Control-P14 and Pou2f2-P14 (the method was the same as 4 and 8 of Example 1). The cells obtained after 24 hours of infection were infused into the tail vein of B6 mice respectively. The infusion method was as follows:

[0230] 6-8 week old B6 mice weighing 20-25 g were divided into two groups: a control group (5 mice) and a Pou2f2 group (5 mice). The mice were infected with LCMV clone 13 (the method was the same as in Example 2).

[0231] Control group: Control-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Control group via tail injection;

[0232] Pou2f2 group: Pou2f2-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2 group via tail injection;

[0233] On the 7th, 14th, 21st and 28th day after transfusion, the percentage of Control-P14 cells and Pou2f2-P14 cells in the peripheral blood of each mouse was analyzed by anti-CD8 antibody and GFP fluorescence flow cytometry. + The proportion of T cells, i.e., the proliferation, contraction, and persistence of the infused P14 cells were monitored in the blood.

[0234] The results are shown in Figure 1C and Figure 1D. During chronic infection with LCMV clone 13, P14 cells in both the control group and the POU2F2-overexpressing P14 cell group (Pou2f2 group) expanded in the blood on day 7 after infection. Subsequently, on days 14 and 21 after infection, the number of P14 cells in the control group gradually decreased and entered the contraction phase, while overexpression of POU2F2 inhibited the decrease in P14 cells during the contraction phase. Until day 28 after infection, P14 cells in the Pou2f2 group remained at the expansion phase level. This indicates that overexpression of POU2F2 inhibits T cells from entering the contraction phase and enhances the persistence of P14 cells and the formation of memory P14 cells.

[0235] 30 days after infection, we analyzed the viral titer of the serum of each group of mice, as shown in Figure 1E:

[0236] Compared with the control group, P14 cells overexpressing POU2F2 better controlled the replication of LCMV clone 13 virus, indicating that P14 cells overexpressing POU2F2 have antiviral efficacy in mice chronically infected with LCMV clone 13.

[0237] Example 5: Overexpression of POU2F2 mutants also significantly enhanced CD8 + T cell expansion and persistence

[0238] Cell reinfusion process: from Cas9 + CD8 cells were isolated from spleen and lymph nodes of P14 transgenic mice + T cells were activated with peptide gp33-41 for 24 hours to obtain activated CD8 + T cells (the method is the same as that of Example 1, step 2); the retrovirus obtained by transfection with pMIGW-GFP, the retrovirus obtained by transfection with pMIGW-POU2F2-GFP, the retrovirus obtained by transfection with pMIGW-POU2F2-184A 185A-GFP, the retrovirus obtained by transfection with pMIGW-POU2F2△281-340aa-GFP, and the retrovirus obtained by transfection with pMIGW-POU2F2-335A339A-GFP were respectively infected with activated CD8+ The recombinant cells obtained were named Control-P14, Pou2f2-P14, Pou2f2-184A 185A-P14, Pou2f2-△281-340aa-P14, and Pou2f2-335A339A-P14 cells (the method was the same as 4-8 of Example 1). The above cells obtained after 24 hours of infection were subjected to flow cytometric analysis. As shown in Figure 1B, compared with the control pMIGW-GFP, T cells transfected with the pMIGW-POU2F2-GFP virus highly expressed POU2F2. The cells were infused into B6 mice through the tail vein, and the specific infusion method was as follows:

[0239] 6-8 week old B6 mice weighing 20-25 g were divided into two groups: control, Pou2f2, Pou2f2-184A 185A, Pou2f2-Δ281-340aa, and Pou2f2-335A 339A, with 5 mice in each group. Mice were infected with LCMV clone 13 (using the same method as in Example 2).

[0240] Control group: Control-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Control group via tail injection;

[0241] Pou2f2 group: Pou2f2-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2 group via tail injection;

[0242] Pou2f2-184A 185A group: Pou2f2-184A 185A-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2-184A 185A group via tail transfusion;

[0243] Pou2f2-△281-340aa group: Pou2f2-△281-340aa-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2-△281-340aa group via tail transfusion;

[0244] Pou2f2-335A 339A group: Pou2f2-335A 339A-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2-335A 339A group via tail transfusion;

[0245] On the 7th, 14th, 21st, 28th and 56th day after transfusion, the percentage of Control-P14 cells, Pou2f2-P14 cells, Pou2f2-184A 185A-P14 cells, Pou2f2-△281-340aa-P14 cells and Pou2f2-335A 339A-P14 cells transfected with empty vector virus in the peripheral blood of each mouse was analyzed by anti-CD8 antibody and GFP fluorescence flow cytometry. + The proportion of T cells, i.e., the proliferation, contraction, and persistence of the infused P14 cells were monitored in the blood.

[0246] The results are shown in Figure 2A and Figure 2B. During chronic infection with LCMV clone 13, on the 7th day after infection, the proliferation levels of the control, POU2F2-overexpressing, and POU2F2-184A 185A P14 cells in the blood were similar, while the proliferation of 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 group gradually decreased and entered the contraction phase, while overexpression of POU2F2 or its several mutants inhibited the decrease of P14 cells in the contraction phase; until 56 days after infection, the P14 cells in the control group shrank to about 1%, while the P14 cells overexpressing POU2F2 or its several mutants still maintained a high proportion, among which the proportion of POU2F2-335A 339A P14 cells overexpressing POU2F2 was the highest. These results indicate that overexpression of POU2F2 or several mutants inhibited the entry of T cells into the contraction phase and enhanced the persistence of P14 cells and the formation of memory P14 cells.

[0247] Example 6: Overexpression of POU2F2 significantly enhanced CD8 + T cell expansion and persistence

[0248] The cell reinfusion process is shown in Figure 3A: + CD8 cells were isolated from spleen and lymph nodes of P14 transgenic mice + T cells were activated with peptide gp33-41 for 24 hours to obtain activated CD8 + T cells (the method is the same as that of Example 1, step 2); the retrovirus obtained by transfection with pMIGW-GFP and the retrovirus obtained by transfection with pMIGW-POU2F2-GFP were respectively infected with activated CD8 +The recombinant cells were named Control-P14 and Pou2f2-P14 (the method was the same as 4 and 8 of Example 1). The cells obtained after 24 hours of infection were infused into the tail vein of B6 mice respectively. The infusion method was as follows:

[0249] 6-8 week old B6 mice weighing 20-25 g were divided into two groups: a control group (5 mice) and a Pou2f2 group (5 mice). LCMV Amstrong infected mice (the method was the same as in Example 2).

[0250] Control group: Control-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Control group via tail injection;

[0251] Pou2f2 group: Pou2f2-P14 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2 group via tail injection;

[0252] On the 7th, 14th, 21st and 28th day after transfusion, the percentage of Control-P14 cells and Pou2f2-P14 cells in the peripheral blood of each mouse was analyzed by anti-CD8 antibody and GFP fluorescence flow cytometry. + The proportion of T cells, i.e., the proliferation, contraction, and persistence of the infused P14 cells were monitored in the blood.

[0253] The results are shown in Figure 3B and Figure 3C. During acute infection with LCMVA mstrong, on day 7 after infection, P14 cells in both the control group and the POU2F2-overexpressing P14 cell group (Pou2f2 group) expanded in the blood, and overexpression of POU2F2 enhanced the expansion of P14. Subsequently, on days 14, 21, and 28 after infection, the number of P14 cells in the control group gradually decreased and entered the contraction phase, while overexpression of POU2F2 significantly inhibited the decrease in P14 cells during the contraction phase. This indicates that overexpression of POU2F2 inhibits T cells from entering the contraction phase and enhances the persistence of P14 cells.

[0254] Example 7: Overexpression of POU2F2 significantly enhanced CD8 + T cell expansion and persistence

[0255] The cell reinfusion process is shown in Figure 4A: + Isolation of CD8 T cells from spleen and lymph nodes of OT1 transgenic mice +T cells were activated with the polypeptide OVA257-264 for 24 hours to obtain activated OT1 cells (the method is the same as in Example 1, step 3); the retrovirus obtained by transfection with pMIGW-GFP and the retrovirus obtained by transfection with pMIGW-POU2F2-GFP were then used to infect the activated OT1 cells, respectively, to obtain recombinant cells named Control-OT1 and Pou2f2-OT1 cells (the method is the same as in Example 1, steps 9-10). The cells obtained by infection for 24 hours were infused into the tail vein of B6 mice, and the infusion method was as follows:

[0256] 6-8 week old B6 mice weighing 20-25 g were divided into two groups: a control group (5 mice) and a Pou2f2 group (5 mice). LM-OVA was used to infect the mice (the method was the same as in Example 2).

[0257] Control group: Control-OT1 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Control group via tail injection;

[0258] Pou2f2 group: Pou2f2-OT1 cells prepared according to the method of Example 1 were prepared into a cell suspension with PBS and then transfused into each mouse in the Pou2f2 group via tail injection;

[0259] On the 7th, 14th and 28th day after transfusion, the percentage of Control-OT1 cells and Pou2f2-OT1 cells in the peripheral blood of each mouse was analyzed by anti-CD8 antibody and GFP fluorescence flow cytometry. + The proportion of T cells, i.e., the proliferation, contraction, and persistence of infused OT1 cells were monitored in the blood.

[0260] The results are shown in Figure 4B and Figure 4C. During LM-OVA infection, OT1 cells in both the control group and the POU2F2-overexpressing P14 cell group (Pou2f2 group) expanded in the blood on day 7 after infection, and overexpression of POU2F2 significantly enhanced OT1 expansion. Subsequently, on days 14 and 28 after infection, the number of OT1 cells in the control group gradually decreased and entered the contraction phase, while overexpression of POU2F2 significantly inhibited the decrease in OT1 cells during the contraction phase. This indicates that overexpression of POU2F2 inhibits T cells from entering the contraction phase and enhances the persistence of OT1 cells.

[0261] Nucleotide sequence of vector pMIGW-POU2F2-GFP (SEQ ID NO: 1):

[0262] Among them, the single underlined part is EGFP; the double underlined part is the coding sequence of POU2F2, that is, SEQ ID NO: 9.

[0263] The nucleotide sequence of POU2F2 (human) is SEQ ID NO: 11:

[0264] The amino acid sequence of POU2F2 (human) is SEQ ID NO: 12:

[0265] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0266] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

Use of any of the following: POU2F2 protein or its mutants, nucleic acid molecules encoding POU2F2 protein or its mutants, or its promoters: (A) Use in the preparation of products for preventing and / or treating infectious diseases and / or diseases and / or symptoms related to infection; (B) Use in the preparation of products for enhancing the anti-infection ability of immune cells; (C) Use in the preparation of products 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 more than 80% sequence identity, 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 more than 80% sequence identity, 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 POU2F2 protein or promote 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; POU2F2 protein precursor protein or conjugate or complex that can be converted into POU2F2 protein in vivo.

3. The use according to claim 1 or 2, characterized in that, 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. The 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. Use according to any one of claims 1 to 4, characterized in that The disease and / or symptom related to the infection is one or more selected from the group consisting of: Pathological damage caused by the infection; immune cell exhaustion after the infection, including reduced proliferation ability 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 (ILCs), and chimeric antigen receptor NK cells (CAR-NK cells); Optionally, the T cells include at least one of 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; Optionally, the T cells include activated T cells; Optionally, the T cells include 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 the non-recombinant immune cell; Optionally, 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); 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 include activated T cells; Optionally, the T cells include 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.

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 related to the infection.

10. A pharmaceutical composition or a kit, which comprises: (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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