Use of LARP4 and inhibitor lipep thereof in preparation and screening of drug for treating autoimmune and allergic diseases

Abstract

The present invention relates to the technical field of polypeptide drugs, and in particular to, use of LARP4 and an inhibitor LIPEP thereof in the preparation and screening of a drug for treating autoimmune and allergic diseases. According to the technical solution, the research finds that LARP4 is an important checkpoint regulatory factor of a naive CD4+T cell exiting from quiescence, the relationship between RNA stability and CD4+T cell steady-state / adaptive activation is disclosed, and the potential of LARP4 as a preventive and therapeutic target for the autoimmune and allergic diseases is demonstrated. According to an action mechanism of LARP4, in this solution, a competitive peptide inhibitor LIPEP of LARP4 is designed, LIPEP processing can perfectly simulate a deletion state of LARP4, and the severity of the autoimmune and allergic diseases is alleviated in a corresponding mouse model. The technical solution can solve the technical problem in the prior art of lacking drugs capable of modulating T-cell responses and providing therapeutic intervention for related diseases.

Description

Application of LARP4 and its inhibitor LIPEP in the preparation and screening of drugs for the treatment of autoimmune and allergic diseases Technical Field

[0001] This invention relates to the field of peptide drug technology, specifically to the application of LARP4 and its inhibitor LIPEP in the preparation and screening of drugs for the treatment of autoimmune and allergic diseases. Background Technology

[0002] T lymphocytes are crucial for adaptive immune responses against pathogens and tumors. Mature T cells exist in an inactive state (G0 phase) in the blood and peripheral lymphoid organs, characterized by small cell size and low metabolic activity, thus preventing their effector function in response to persistent signals such as self-antigens and other mediators. This state, known as the quiescent state, prevents sporadic expansion of naïve T cells and maintains their relative abundance. The molecular mechanisms controlling the quiescent state also set the threshold for naïve T cell activation. The regulation of the quiescent state is coordinated by a diverse set of transcription factors, including forkhead box (FOX) proteins, Kruppel-like factors (KLFs), members of the APRO (Tob1) family, BTG1 / 2, and the T cell activation inhibitory factor (VISTA) containing a type V immunoglobulin domain.

[0003] Nascent T cells are maintained in a quiescent state, an inactive state, and their exit from this state is a marker of antigen stimulation. Quiescent state exit is a transitional state in the T cell activation process, occurring between antigen recognition and co-stimulatory signaling, and active proliferation. Quiescent state exit affects T cell proliferative capacity, metabolic adaptation, differentiation, and effector function. Markers of nascent T cell exit from the quiescent state include entering the cell cycle, interleukin-2 (IL-2) production, cell growth, and anabolism. Research into the molecular mechanisms underlying quiescent state exit can pave the way for regulating T cell responses and implementing therapeutic interventions, including interventions for autoimmune diseases, infectious diseases, and cancer.

[0004] T cell activation plays a crucial role in the immune system's fight against external infections and other types of diseases. Under homeostasis, T cells remain in a resting state, but abnormal activation can lead to disease. Although maintaining the resting state of T cells is essential, the underlying mechanisms remain poorly understood. In-depth research into the molecular mechanisms of T cell resting state maintenance and de-resting is urgently needed to develop drugs that regulate T cell resting state, as well as drugs for autoimmune diseases, infectious diseases, and cancer.

[0005] Summary of the Invention

[0006] The present invention aims to provide a LARP4 inhibitor, LIPEP, to address the technical problem of the lack of drugs in the prior art that regulate T cell responses and provide therapeutic intervention for related diseases.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] LIPEP, an LARP4 inhibitor, is used to block the interaction between LARP4 and PABP.

[0009] Furthermore, LARP4 interacts with the MLLE domain of PABP through the PAM2 motif.

[0010] Furthermore, the amino acid sequence of the PAM2 motif of LARP4 is shown in SEQ ID NO.2; the amino acid sequence of the MLLE domain of PABP is shown in SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of LIPEP is shown in SEQ ID NO.1.

[0012] This technical solution also provides the application of the LARP4 inhibitor LIPEP in the preparation of drugs for treating or preventing diseases caused by abnormal T cell activation or T cell dysfunction. This technical solution provides a LIPEP peptide that can inhibit the binding of LARP4 to PABP, thereby regulating the T cell resting state (regulating the exit of naive T cells from the resting state), thus achieving the treatment or prevention of diseases caused by abnormal T cell activation or T cell dysfunction. Notably, LARP4 is associated with the exit of naive T cells (Tn, also known as unsensitized T cells) from the resting state, a concept first proposed in this solution, providing a new target for drug action mechanism research, and leading to the development of the novel peptide inhibitor LIPEP.

[0013] Furthermore, diseases caused by abnormal T cell activation or T cell dysfunction include autoimmune diseases and allergic diseases.

[0014] Furthermore, the autoimmune diseases mentioned include multiple sclerosis.

[0015] Furthermore, the allergic diseases mentioned include asthma.

[0016] This technical solution also provides the application of LARP4 in screening or designing drugs that regulate the exit of naive T cells from the resting state. By analyzing the interaction between the PAM2 motif of LARP4 and the MLLE domain of PABP or PABPC, this technical solution screened for the LARP4 inhibitor LIPEP, which could serve as a drug for diseases related to the exit of naive T cells from the resting state. LARP4 is likely the primary therapeutic target for LIPEP.

[0017] Furthermore, drugs that regulate the exit of naive T cells from the resting state include competitive peptide inhibitors that inhibit the interaction between the PAM2 motif of LARP4 and the MLLE domain of PABP, or systems that inhibit the expression of the LARP4 gene or protein. Drugs used to treat or prevent diseases associated with the exit of naive T cells from the resting state interact with the PAM2 motif of LARP4, thereby blocking the interaction between LARP4 and PABP or between LARP4 and PABPC. Additionally, experimental results have shown that inhibiting the expression of the LARP4 gene or protein (e.g., gene knockout) can also enhance the resting state and attenuate the exit of naive T cells from the resting state.

[0018] In summary, the principle and beneficial effects of this technical solution are as follows:

[0019] T cell exit from the resting state is a key checkpoint in the T cell journey, and elucidating the underlying molecular mechanisms has been a long-standing goal of immunological research. While several major transcriptional regulators (such as Klf2 and Foxp1) have been identified, our understanding of post-transcriptional regulation remains limited. This protocol demonstrates the role of T cells in the initial CD4+ ionotropic response. + Conditional knockout of LARP4 in T cells resulted in attenuated resting-state exit and / or clonal expansion. Furthermore, evidence suggests that LARP4 is essential for robust helper T cell differentiation and antigen-specific immune responses. Conversely, LARP4 deficiency may be beneficial under autoimmune and allergic conditions, as LARP4 CKO alleviated disease progression in mouse EAE and HDM models. Therefore, this protocol proposes for the first time LARP4 as a key immunomodulatory factor for fine-tuning immune response robustness (Figure 29) and potentially as a target for the prevention and treatment of autoimmune and allergic diseases.

[0020] LARP4, a member of the La-associated protein (LARP) family, protects mRNA from degradation by stabilizing the poly(A) tail's PABP. LARP4 can directly bind to both polyA and PABP, protecting the poly(A) tail length, and is associated with mRNA stabilization. In this study, LARP4 was identified as a novel positive / stimulatory regulator for exiting the quiescent state.

[0021] LIPEP (LARP4 inhibitory peptide) can enter the initial CD4 in vitro. +T cells were stimulated to competitively inhibit LARP4-PABPC1 interaction in a dose-dependent manner. LIPEP entered cells at high concentrations, although the molecular mechanism by which the LIPEP peptide crosses the plasma membrane into the cytoplasm remains unclear. Based on existing knowledge of cell-penetrating peptides (CPPs), it is speculated that the N-terminus of LIPEP is amphiphilic, possessing both polar and nonpolar regions, which facilitates its entry into cells. N-terminal palmitoylation of LIPEP increases the nonpolar lipophilic group, making it easier for LIPEP to bind to the cell membrane. Furthermore, the predominantly positive surface charge of the helical structure at the N-terminus of LIPEP may induce transient pores in the lipid bilayer, allowing LIPEP to enter the cytoplasm. Regarding T cell activation and differentiation, LIPEP-treated naïve CD4+... + T cell manifestations and initial CD4 after conditional knockout of LARP4 + T cells are similar. In EAE and HDM-induced allergic disease models, T cells are continuously activated, suggesting that the therapeutic effect of LIPEP in vivo may be due to the inhibition of sustained T cell activation. Data indicate that LARP4 may serve as a therapeutic target for autoimmune diseases, and developing competitive peptide inhibitors of LARP4 is a novel approach for related drug development. Compared to other proteins containing PAM2, LARP4 is the primary target of LIPEP. LIPEP treatment mimics the LARP4CKO phenotype in several mouse disease models, and overexpression of LARP4 can functionally rescue LIPEP-treated T cells. Therefore, LIPEP primarily functions through LARP4 as a downstream effector.

[0022] The beneficial effects of this technical solution are as follows:

[0023] (1) The study discovered the molecular mechanism by which LARP4 affects T cell resting state exit, and confirmed that LARP4 can serve as a therapeutic target for diseases related to T cell resting state exit, thus laying the foundation for related drug development.

[0024] (2) The interaction mechanism between LARP4 and PABP or PABPC was studied in depth, and LIPEP (a competitive peptide inhibitor of LARP4) was designed based on the MLLE domain of PABP. LIPEP has a strong affinity for LARP4 and can block the interaction between LARP4 and PABP or PABPC, thereby achieving the treatment of T cell resting state withdrawal related to the disease. Attached Figure Description

[0025] Figure 1 shows the preparation of Larp4 conditional knockout mice in Example 1 (A: Larp4). fl / flA schematic diagram of the construction of Cd4-cre mice (CKO). These transgenic mice were prepared by BIOCYTOGEN Technology Ltd. (Beijing, China); B: WB shows CD4 from CKO, KO, and WT mice. + Expression of LARP4 protein in T cells.

[0026] Figure 2 shows the results of the study on the interaction between LARP4 and the resting state of naive T cells in Example 1 (A: Frequency of CD4 single-positive (CD4 SP) T cells from mouse thymus determined by flow cytometry; B: Frequency of CD4 single-positive (CD4 SP) T cells from mouse spleen determined by flow cytometry). + T cells CD62L + CD44 - (Initial) or CD62L - CD44 + (Effective) T cell frequency; C: Cell cycle analysis using 4',6-diamino-2-phenylindole (DAPI) and Ki-67 staining, and initial CD4+. + D: Percentage of Ki-67 positive cells in T cells; E: Size and granularity of control or CKO naïve T cells measured by flow cytometry (MFI = mean fluorescence intensity); F: Initial CD4 count measured by Western blotting and real-time quantitative PCR (RT-qPCR). + The relative expression levels of Klf2, Klf6, and Foxp1 genes in T cells, with GAPDH or β-tubulin used as internal controls; FH: in vitro T cell proliferation assay; in F, naïve CD4 + T cells were co-cultured for 4 days with anti-CD3 (0, 1.25, 2.5, and 5.0 μg / ml) and anti-CD28 (1 μg / ml) antibodies fixed on a plate. A representative dot plot shows the initial CD4 cells from the control group and CKO mice. + The production of IL-2 and Ki-67 in T cells; in G, cell division was represented by carboxyfluorescein succinimide (CFSE) labeling, with populations >1 division represented by mean ± standard error (SEM); in H, flow cytometry showed cell surface expression of CD25, CD69, CD122, and CD44; in I, CD44 levels in the spleen of control and CKO mice on day 8 post-LCMV infection. + GP66 + CD44 + Absolute T cell count; in J, in recipient mice (CD45.1) + CD45.2 was examined in the spleen. + GP66 + CD4 + CD45.2+ GP66 + CD4 + CD44 + And CD45.2 + GP66 + CD4 + CD69 + The absolute number of T cells (CKO mice), measured one day prior to infection from iLARP4 - / - (CD45.2 + ) and Larp4 + / + (CD45.2 + 2 × 10⁻⁶ mice were selected. 4 Initial CD4 + T cells were transferred to recipient mice (CD45.1) after treatment with tamoxifen on days 0, 1, 2, and 3. + In the K group, mice were subsequently infected with LCMV, and the absolute number of indicator cells was determined by flow cytometry on day 8 post-infection. In the K group, the concentrations of LCMV-specific IgM and IgG were measured by ELISA from the serum of control and CKO mice on day 8 post-LCMV infection (D8). Representative data in the AK group were from three independent experiments, n = 5 mice per group. AK data are shown as mean ± SEM. NS = not significant, *P < 0.05, ***P < 0.001, by unpaired t-test. C57BL / 6J mice were between 6 and 10 weeks old, with no sex preference.

[0027] Figure 3 shows the effect of LARP4 conditional knockout on apoptosis or cell death in Example 1 (flow cytometry analysis of control group and CKO spleen with initial CD4). + T cells; annexin V and 7-AAD staining; n = 5 mice per group, data are expressed as mean ± SEM; NS = not significant).

[0028] Figure 4 shows the validation of T cell activation-related proteins in Example 1 (the protein levels of T cell activation-related genes, including TCRβ, CD3ε, CD2, pERK, NUR77 and pZAP70 (n=3 per group), were measured by flow cytometry; data are expressed as mean ± SEM; NS = not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0029] Figure 5 shows the verification results of LARP4 re-expression in CKO cells in Example 1 (A: WB shows CD4 transduced via retroviral empty vector (EV) or LARP4 vector). +B: Flow cytometry plots show LARP4 protein expression in T cells, with GAPDH serving as an internal control; B: Flow cytometry plots show CD4 expression. + Cell surface expression of CD25, CD69, CD122, and CD44 in T cells; n = 5 mice per group, data are expressed as mean ± SEM; NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0030] Figure 6 shows LARP4 and the initial CD8 in Example 1. + Results of a study on the role of resting T cell status (A: Flow cytometry was used to determine CD8+ from the spleen of a specified mouse cell). + CD62L in T cells + CD44 - (Initial) or CD62L - CD44 + (Effective) T cell frequency; B: CD8 count in control group or CKO initial CD8 measured by flow cytometry. + T cell size and granularity (MFI = mean fluorescence intensity); C and D: In vitro T cell proliferation assay, initial CD8 + T cells were co-cultured for 4 days with anti-CD3 (0, 1.25, 2.5, and 5.0 μg / ml) and anti-CD28 (1 μg / ml) antibodies fixed on a plate. A representative dot plot shows the initial CD4 cells from the control group and CKO mice. + The production of IL-2 and Ki-67 by T cells; flow cytometry images showed the cell surface expression of CD25, CD69, CD122, and CD44; E: CD8+ expression in the spleen of control and CKO mice on day 8 after LCMV infection. + GP33 + CD44 + Absolute T cell count; representative data in AE were from three independent experiments, n = 5 mice per group, and data are expressed as mean ± SEM; NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

[0031] Figure 7 shows the transcriptome results of the control group and CKO mice in Example 1 (A: showing the initial CD4 from the control group and CKO mice). + A plot of total RNA-seq reads from T cells, with dots representing upregulated genes (red), downregulated genes (blue), and unchanged genes (gray), and dashed lines indicating differences of ±0.263; B: Gene set enrichment analysis (GSEA) pathway enrichment plot, showing the initial CD4 counts in CKO and control groups. +Representative gene sets enriched in T cells, each gene set showing a normalized enrichment score (NES) and p-value calculated using the Kolmogorov-Smirnov test; C: Classification of LARP4-bound endogenous RNAs revealed by RIP-seq; D: Top 20 most significant GO terms for LARP4-bound protein-coding RNAs, gene ratios referring to the ratio of observed gene counts to background gene counts; E: Schematic diagram of the RNA decay assay protocol, from initial CD4 in mice. + T cells were treated with flavopiridol (FLV), and cells were collected at 0 or 2 hours later. Total RNA was subjected to deep sequencing analysis; F: Scatter plot showing the control group and CKO initial CD4. + Gene degradation rates in T cells, with dashed lines indicating differences of ±0.1; G: Venn diagram showing overlapping genes of LARP4-binding target genes showing decreased mRNA levels and increased degradation rates after LARP4 CKO; H: Top 20 most significant GO and KEGG pathways for protein-coding RNA among 158 overlapping genes.

[0032] Figure 8 shows the LARP4 binding event enrichment site analysis of Example 1 (LARP4-mRNA interaction events are enriched at the distal end of the 3'UTR; the binding profile is derived from ENCODE LARP4 eCLIP-Seq data of K562 and HepG2 cells; the y-axis represents the density of LARP4 binding events).

[0033] Figure 9 shows the verification of the poly(A) tail length of Cd3e, Cd2, Cd44, Icos, and Batf mRNA in Example 1 (controlling initial CD4). + Blurred bands in the poly(A) PCR products of T cells are indicated by asterisks; the corresponding bands of CKO cells are positioned on the gel to show differences; RT+, reverse transcription; RT-, no reverse transcription; Poly(A) tail PCR products were also ligated into the pZeroback Blunt vector, and the resulting clones were sequenced by Sanger sequencing to determine the actual length of the poly(A) tail; scatter plots show the differences in poly(A) tail lengths of Cd3e, Cd2, Cd44, Icos, and Batf mRNA between blank control (Ctrl) and CKO cells; horizontal lines and error bars represent the mean ± SD of all sequenced clones; *P < 0.05 ***P < 0.001; ****P < 0.0001).

[0034] Figure 10 shows the effect of LARP4 on the differentiation of Th1 / Th2 / Th17 cells in vitro and in vivo in Example 1 (AC: CD4 from mouse spleen). + CD4 in T cells+ IL - 17A + Th17, CD4 + IFN - γ + Th1 and CD4 + IL - 4 + Frequency of Th2 cells, representative cytokines and transcription factors; D: CD4 from indicator mouse spleen + CD4 in T cells + FOXP3 + Frequency of Treg cells, representative surface markers, and transcription factors; E: Unsupervised clustering was performed on t-SNE to identify cell populations with similar gene expression, and bubble plots show the mean Z-transform normalized expression of typical immune cell markers in each cluster; F: t-SNE plots were used to identify cell populations in control and CKO mice; G: CD4 + Cellular composition of T cell subsets; H: primate and effector CD4 + Percentage of T cell population; IK: processed initial spleen CD4 + T cells were stimulated for 5 days under Th17, Th1, and Th2 polarization conditions, and CD4 counts were measured by flow cytometry. + The frequency of T cell subsets was measured, and the concentrations of representative cytokines in cell culture supernatant were determined by ELISA. Representative data were obtained from three independent experiments, with n = 5 mice in each group. Data are expressed as mean ± SEM. NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0035] Figure 11 shows the intracellular staining results of IFN-γ, IL-4, and IL-17 in CFSE-labeled T cells cultured under Th1 / 2 / 17 conditions in Example 1 (AC: initial CD4+ of CFSE-labeled cells). + T cells were stimulated for 5 days under Th1, Th2, and Th17 polarization conditions, and CD4 counts were measured by flow cytometry. + Frequency of T cell subsets; D: On day 5 of polarization, an equal volume of live control or CKO cells were stimulated again with anti-CD3 / anti-CD28, and the supernatant was then collected and cytokines were assessed by ELISAs; n = 5 mice per group, data are expressed as mean ± SEM).

[0036] Figure 12 shows the effect of LARP4 deficiency on autoimmune encephalomyelitis and allergic asthma in Example 1 (A: in Cd4-cre (control group) and LARP4 deficiency). fl / flA schematic diagram of the EAE disease model established in Cd4-cre (CKO) mice, showing the mean daily clinical scores of the control group and CKO mice after EAE induction (two-way ANOVA); B: Spinal cord samples collected from mice on day 30 after EAE induction were stained with hematoxylin and eosin (HE) or Lugol's fast blue (LFB) to assess inflammation and myelin content, respectively. Outlines indicate inflammatory or demyelinated lesions. Scale bar: 200 or 100 micrometers; C: CD4+ in leukocytes isolated from the spinal cord of mice on day 30 after EAE induction. + T cells were further analyzed to determine CD4. + T cell frequency and absolute number; D: CD4 count in leukocytes isolated from the spinal cord of mice on day 30 after EAE induction. + T cells were further analyzed by flow cytometry to determine CD4. + IL - 17A + Frequency of Th17 cells; E: Schematic diagram of house dust mite (HDM) allergy model established in control and CKO mice; F: Total number of cells and number of eosinophils in BAL fluid measured by flow cytometry after HDM challenge; GH: Representative lung sections and cumulative total inflammation (H&E) and mucus (AB-PAS) scores; ip = intraperitoneal, it = intratracheal, sc = subcutaneous; Representative data from three independent experiments, n = 5 mice per group; Data are expressed as mean ± SEM; NS = not significant, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

[0037] Figure 13 shows the results of the study on the correlation between the reduction of EAE severity in LARP4-deficient mice and Th17 cells in Example 1 (AC: CD4+ in leukocytes isolated from mouse spinal cord on day 30 after EAE induction). + T cells were further analyzed to identify CD4. + IFN - γ + Th1, CD4 + IL - 4 + Th2 and CD4 + FOXP3 + Frequency of Treg cells; D: Monocytes collected from drained lymph nodes on day 8 and further compared in vitro with MOGs. 35-55 After co-culturing for 3 days, the concentrations of IL-17A, IFN-γ, and IL-4 were measured by ELISA; representative data were obtained from three independent experiments, n = 5 mice per group, and data are expressed as mean ± SEM; NS = not significant, *P < 0.05 and **P < 0.01).

[0038] Figure 14 shows MOG-specific CD4 in the EAE model of Example 1. + Results of a study on T cell initiation and cytokine production (AB: mononuclear cells collected from draining lymph nodes on day 8 and further analyzed in vitro with MOG). 35-55 After co-culturing for 3 days, CD4 cells were isolated. + Further analysis of T cells was conducted to determine I-Ab MOG. 35-55 Tetramer + CD4 + T cell frequency, then I-Ab MOG isolation 35-55 Tetramer + CD4 + T cells were used to determine the mean fraction (MFI) of IL-17A and IFN-γ (n = 6 mice per group); mean ± SEM is shown; NS = not significant, *P < 0.05).

[0039] Figure 15 shows the design and characteristics of LIPEP in Example 2 (A: Schematic diagram illustrating LIPEP's blocking of LARP4-mediated mRNA stability; B: Key position of the MLLE domain and its effect on interactions with PAM2-containing proteins; CD: Conformation of the key position P10 in the PAM2 motif; EF: Hydrophobic interaction surfaces of LIPEP(e) and MLLE(f); GH: Schematic diagram of the 2D interaction between the PAM2 motif of LARP4 and LIPEP(g) and MLLE(h); I: Calculation of the interaction between LIPEP / MLLE and the PAM2 motif using CHARMm and DS2.5 tools). Interaction energy; JK: Relative levels of LIPEP binding to various PAM2-containing proteins, n=3 per group, data are expressed as mean ± SEM; L: Data and graphs show plasma LIPEP concentrations (ng / mL) in ICR (CD-1) mice after subcutaneous injection of 100 mg / kg body weight of LIPEP per mouse, data are expressed as mean ± standard deviation (n=3), hr = hours; M: Pharmacokinetic parameters of LIPEP: half-life (T1 / 2), maximum concentration at a specific time (Tmax) (Cmax), area under the curve (AUC0-∞), mean residence time (MRT0-∞)).

[0040] Figure 16 shows the results of the mouse toxicity study of LIPEP in Example 2 (A: Representative tissue sections of liver, kidney, intestine, lung, spine, brain, spleen and heart stained with Hematoxylin & Eosin in mice in the control group and mice treated with 100 mg / kg LIPEP; control group mice were treated with 2% mannitol; B: Comparison of serum total protein (TP), albumin (ALB), direct bilirubin test (TBIL), glucose (GLU), alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GT), aspartate aminotransferase (AST), alanine aminotransferase test (ALT), urea (UREA), carbamate (CREA), creatine kinase (CK), cholesterol (CHOL) and triglycerides (TG) in different groups of mice, n = 7 mice in each group, data are expressed as mean ± SEM; NS = not significant, *P < 0.05, **P < 0.01).

[0041] Figure 17 shows the results of the study on the effect of LIPEP on apoptosis or cell death in Example 2 (flow cytometry analysis of wild-type (WT) spleen naïve CD4+ T cells after LIPEP treatment, stained with annexi V and 7-ADD; n = 5 mice per group, data are expressed as mean ± SEM; NS = not significant, *P < 0.05).

[0042] Figure 18 shows the results of the study on the relationship between LIPEP and the mRNA stability and expression of genes related to resting-state exit in Example 2 (A: Confocal fluorescence microscopy results, scale bar: 5 μm; B: Flow cytometry results; C: Immunoprecipitation results; D: RIP-qPCR verification of the relationship between LARP4 and Cd2, Cd3e, Cd44, Icos, and Batf mRNAs after LIPEP treatment in the initial CD4+). + Changes in T cell interactions, with Ct values ​​normalized to the Ct values ​​of GAPDH mRNA not bound by LARP4; E: Initial CD4 counts after LIPEP treatment were determined by label-free RT-qPCR. + Changes in the stability of Cd3e, Cd2, Cd44, Icos, and Batf mRNAs in T cells were observed after FLV treatment for 0, 1, and 2 hours, with GAPDH used as an internal control; F: Initial CD4 counts after LIPEP treatment were measured by RT-qPCR. +The relative expression changes of Cd2, Cd3e, Cd44, Icos, and Batf mRNAs in T cells, with GAPDH used as an internal control; representative data were obtained from three independent experiments, n = 5 mice per group, and data are expressed as mean ± SEM; NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 or ####P < 0.001 and #####P < 0.0001).

[0043] Figure 19 shows the LIPEP in Example 3 on the initial CD4. + Experimental results of simulating LARP4 deficiency in T cells (A: Initial CD4 count was measured by RT-qPCR 8 hours after treatment with different concentrations of LIPEP). + The relative expression of Klf2, Klf6, and Foxp1 mRNAs in T cells, with GAPDH as an internal control; B: naïve CD4 + T cells were treated with different concentrations of LIPEP, and the cell cycle was analyzed by flow cytometry 8 hours after the addition of LIPEP; C: naïve CD4 + T cells were stimulated with anti-CD3 / CD28 antibody, and then LIPEP was added at different time points or at different concentrations. Twenty-four hours after LIPEP addition, cell cycle analysis of the naïve T cells was performed by flow cytometry using Ki-67 and DAPI staining. D: Naïve CD4 cells under different time points with LIPEP addition. + T cells were stimulated under Th17 polarization conditions, and CD4 counts were measured by flow cytometry. + IL - 17A + Frequency of Th17 cells; E: Initial CD4 count under different time points with LIPEP administration + T cells were stimulated under Th1 polarization conditions, and CD4 counts were measured by flow cytometry. + IFN - γ + Frequency of Th1 cells; F: Initial CD4 count under different time points with LIPEP administration. + T cells were stimulated under Th2 polarization conditions, and CD4 counts were measured by flow cytometry. + IL - 4 + Frequency of Th2 cells; G: Total 2 × 10 4 An initial CD45.1, either processed or unprocessed with LIPEP. + SMARTA cells were adopted and transferred to naïve wild-type (CD45.2) cells. +In recipient mice, the mice were intraperitoneally infected with LCMV Armstrong strain on day 2. On day 8 post-infection, CD4 counts were assessed in the host spleen by flow cytometry. + CD45.1 + Vα2 + CD45.1 + Vα2 + CD4 + CD44 + And CD45.1 + Vα2 + CD4 + CD69 + Absolute cell number; data are expressed as mean ± SEM, representative data from three independent experiments, n = 5 mice per group; NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

[0044] Figure 20 shows the experimental results of Larp4 mRNA expression level after TCR stimulation in Example 3 (initial CD4 in mice). + T cells were co-cultured with anti-CD3 (2 μg / ml) and anti-CD28 (1 μg / ml) antibodies for 2 days. larp4 mRNA expression at different time points was quantified by RT-qPCR. GAPDH was used as an internal control. Representative data were obtained from three independent experiments. Data are expressed as mean ± SEM; NS = not significant, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

[0045] Figure 21 shows the CD4+ expression after LARP4 overexpression in Example 3 or the LIPEP treatment group. + Expression levels of PAM2 protein, mRNA, and cytokines in T cells (A: Relative expression levels of mRNA encoding PAM2 protein were measured by RT-qPCR, with GAPDH used as an internal control, n=5 per group; B: Relative levels of LARP4 and LARP4B proteins were shown by Western blotting, with GAPDH used as an internal control, n=3 mice per group. Western blotting images were generated using the ChemiDoc XRS system (Bio-Rad), and the levels of indicator proteins were quantified using Image Lab software; C: Relative expression levels of IFN-γ, IL-4, and IL-17A mRNA were determined by RT-qPCR, and the concentrations of IFN-γ, IL-4, and IL-17A were detected by ELISA, n=5 per group; shown as mean ± SEM, NS = not significant, ***P < 0.001).

[0046] Figure 22 shows the effect of LIPEP on inhibiting human CD4 in PBMCs of healthy volunteers in Example 3. + Results of a study on the expression of transcription factors and cytokines in T cell subsets (A: Flow cytometry was used to detect the entry of LIPEP into human CD4 at different concentrations). + T cell status; B: Human CD4 isolated from PBMCs of healthy volunteers. + T cells were treated with different concentrations of LIPEP for 8 hours, followed by anti-CD3-CD28 stimulation for 24 hours. Cells and culture supernatants were then collected. The relative expression of Rorc and Il17a mRNA was determined by RT-qPCR, and the concentration of IL-17A was measured by ELISA. GAPDH served as an internal control. C: The relative expression of Tbx21 and Ifng mRNA was determined by RT-qPCR, and the concentration of IFN-γ was measured by ELISA. GAPDH served as an internal control. D: The relative expression of Gata3 and Il4 mRNA was determined by RT-qPCR, and the concentration of IL-4 was measured by ELISA. GAPDH served as an internal control. Representative data were obtained from three independent experiments (n = 5 healthy donors). Data are expressed as mean ± SEM. NS = not significant, *P < 0.05, **P < 0.01, ****P < 0.0001).

[0047] Figure 23 shows whether LIPEP in Example 4 affects the initial CD4 of CKO. + Experimental results of Cd3e, Klf2 and Klf6 mRNA expression in T cells (A: Initial CD4 expression of CKO cells was determined by RT-qPCR 8 hours after addition of different concentrations of LIPEP). + The relative expression of cd3e mRNA in T cells, with GAPDH as an internal control; B: Initial CD4 expression of CKO cells was determined by label-free RT-qPCR at three time points (0, 1, and 2 hours after FLV treatment). + Changes in the stability of cd3e mRNA in T cells, with GAPDH as an internal control; CD4: Initial CD4 count in CKO cells was determined by RT-qPCR after 8 hours of adding different concentrations of LIPEP. + The relative expression of Klf2 and Klf6 mRNA in T cells, with GAPDH as an internal control; representative data were obtained from three independent experiments, n=5 mice per group, and data are expressed as mean ± SEM; NS = not significant).

[0048] Figure 24 shows the results of Example 4 regarding whether LIPEP inhibits Th1 / 2 / 17 cell differentiation in the absence of LARP4 (A: CKO initial CD4 in the presence of different concentrations of LIPEP). +T cells were stimulated under Th17 polarization conditions, and CD4 counts were measured by flow cytometry. + IL - 17A + Frequency of Th17 cells; B: CKO initial CD4 in the presence of different concentrations of LIPEP. + T cells were stimulated under Th1 polarization conditions, and CD4 counts were measured by flow cytometry. + IFN - γ + Frequency of Th1 cells; C: CKO initial CD4 in the presence of different concentrations of LIPEP + T cells were stimulated under Th2 polarization conditions, and CD4 counts were measured by flow cytometry. + IL - 4 + Th2 cell frequency; representative data were obtained from three independent experiments, n=5 mice per group, and data are expressed as mean ± SEM; NS = not significant).

[0049] Figure 25 shows the experimental results of the preventive effect of LIPEP treatment on EAE mice in Example 5 (A: Flowchart of preventive LIPEP treatment; B: Mean daily clinical scores of mice after EAE induction (two-way ANOVA); C: Spinal cord samples collected from mice on day 30 were stained with HE or LFB to assess inflammation and myelin content, respectively. Outlines indicate lesions of inflammation or demyelination. Scale bar: 200 or 100 micrometers; D: CD4 count in leukocytes isolated from the spinal cord of indicator mice on day 30 after EAE induction). + T cells were gated and further analyzed to identify CD4. + The frequency of T cells was assessed by flow cytometry, which was used to evaluate the total CD4 count in spinal cord infiltration. + Absolute number of T cells; EH: CD4 count in leukocytes isolated from the spinal cord of mice on day 30 after EAE induction. + T cells were further analyzed by flow cytometry to identify CD4. + IL - 17A + Th17, CD4 + IFN - γ + Th1, CD4 + IL - 4 + Th2 and CD4 + FOXP3 + Treg cell frequency; I: Monocytes were collected from draining lymph nodes on day 8 and compared with MOG cells in vitro. 35-55Mice were co-cultured with LIPEP or mannitol for 3 days, and the concentrations of IL-17A, IFN-γ, and IL-4 were measured by ELISA. Representative data were obtained from three independent experiments, n = 5 mice per group. Data are expressed as mean ± SEM. NS = not significant, *P < 0.05 and ****P < 0.0001.

[0050] Figure 26 shows the experimental results of the therapeutic effect of LIPEP treatment on EAE mice in Example 5 (A: Schematic diagram of the therapeutic LIPEP treatment process; B: Mean daily clinical scores of mice after EAE induction (two-way ANOVA); C: Spinal cord of mice collected on day 30 for HE or LFB staining to assess inflammation and myelin content, respectively. Outlines indicate lesions of inflammation or demyelination. Scale bar: 200 or 100 micrometers; D: CD4 count in leukocytes isolated from the spinal cord of mice on day 30 after EAE induction). + T cells were gated and further analyzed to identify CD4. + The frequency of T cells was assessed by flow cytometry, which was used to evaluate the total CD4 count in spinal cord infiltration. + Absolute number of T cells; EH: CD4 count in leukocytes isolated from the spinal cord of mice on day 30 after EAE induction. + T cells were further analyzed by flow cytometry to identify CD4. + IL - 17A + Th17, CD4 + IFN - γ + Th1, CD4 + IL - 4 + Th2 and CD4 + FOXP3 + Frequency of Treg cells; representative data from three independent experiments, n=5 mice per group, data are expressed as mean ± SEM, NS=not significant, *P<0.05).

[0051] Figure 27 shows the experimental results of the preventive effect of LIPEP treatment in an allergic disease mouse model in Example 5 (A: Schematic diagram of LIPEP treatment process; B: Total cell and eosinophil counts in BAL fluid measured by flow cytometry after HDM challenge; C: Representative lung sections and cumulative total inflammation (H&E) and mucus (AB-PAS) scores; Representative data are from three independent experiments, n=5 mice in each group, and data are expressed as mean ± SEM; NS=not significant, *P<0.05, **P<0.01 and ***P<0.001).

[0052] Figure 28 shows the experimental results of the therapeutic effect of LIPEP treatment in an allergic disease mouse model according to Example 5 (A: Schematic diagram of LIPEP treatment process; B: Total cell and eosinophil counts in BAL fluid measured by flow cytometry after HDM challenge; C: Representative lung sections and cumulative total inflammation (H&E) and mucus (AB-PAS) scores; Representative data are from three independent experiments, n=5 mice in each group, and data are expressed as mean ± SEM; NS = not significant and **P < 0.01).

[0053] Figure 29 is a schematic diagram of the initial T cells exiting the resting state.

[0054] Figure 30 shows the results of the peptide screening experiment (Pep-KE) of Comparative Example 1.

[0055] Figure 31 shows the results of the peptide screening experiment (Pep-KD) in Comparative Example 1.

[0056] Figure 32 shows the results of the peptide screening experiment (Pep-RE) of Comparative Example 1. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0058] Example 1: Study on the mechanism of action of LARP4

[0059] (1) LARP4 deficiency enhances the resting state and weakens the exit of naive T cells from the resting state.

[0060] To determine the role of LARP4 in T cell function in vivo, a LARP4 assay was constructed using conventional techniques. fl / fl Mouse strains, and in CD4 + LARP4 was conditionally knocked out in T cells, and the truncated protein (Larp4) was not detected in LARP4 CKO samples. fl / flCd4-cre (CKO, Figure 1). To construct conditional knockout mice, loxP fragments are typically inserted into introns downstream of exons containing ATG. Removing flanking exons results in protein reading frame shifts. After scanning gene structure and exon sizes, it was found that exons 3 (ENSMUSE00000344606) and 4 (ENSMUSE00000489242) could be conditionally removed. The deletion of exons 3-4 caused reading frame shifts, resulting in a premature stop codon (codon 60: 51 original amino acids + 9 extra amino acids) and nonsense-mediated decay (NMD). Gene knockout mice were designed by conditionally knocking out exons 3-4 using the Cre-loxP system. Furthermore, introns 2 and 4 are relatively large, and the insertion of loxP elements does not interfere with mRNA splicing.

[0061] No significant differences in T cell development were detected in the thymus of CKO mice and their littermates (Fig. 2A). However, in the spleen of LARP4 CKO mice, naïve and effector CD4 cells showed significant differences. + The frequency of T cells increased (52.58% vs. 66.64%, p < 0.001) and decreased (18.98% vs. 15.28%, p < 0.05), respectively (Figure 2B). Apoptosis analysis showed that CKO and control naïve CD4 cells... + There was no significant difference in T cell death or apoptosis among them (Figure 3). Detailed analysis showed that the initial CD4 in LARP4-deficient (LARP4 CKO mice) mice... + T cell proliferation was reduced because a significantly higher proportion of cells were in the G0 phase (Figure 2C). In fact, the initial CD4+ of LARP4 CKO mice... + Compared to control naive T cells, T cells showed reduced cell volume and decreased granulation (Figure 2D). Consistent with these findings, the mRNA and protein levels of key resting-state regulators (such as KLF2, KLF6, and FOXP1) were significantly lower in naive CD4+ cells from LARP4 CKO mice. + A significant increase was observed in T cells (Figure 2E). Furthermore, CKO-naïve CD4... + The expression levels of CD3ε, TCRβ, and CD2 proteins on the surface of T cells were also reduced (Figure 4). The early TCR signaling gene Nur77 is present in the initial CD4+ expression of CKO. + The levels of these intermediates in T cells are also reduced (Figure 4), and the phosphorylation signaling intermediates of the TCR, including pERK and pZAP-70, are also reduced (Figure 4), which means that CKO-initiated CD4+ is also reduced. + T cells exhibit only weak TCR signaling. Taken together, these data suggest that LARP4 is responsible for the initial CD4+ signal. + The transformation of T cells into effector cells is necessary and may play an important role in the process of quiescent state withdrawal.

[0062] To determine whether LARP4 affects the initial CD4 + T cell activation was first assessed through in vitro T cell proliferation experiments. Under TCR stimulation, naïve CD4 cells derived from CKO mice were observed by double staining with IL-2 and Ki-67 (Fig. 2F) or by staining with the cell division tracking dye CFSE. + The percentage of proliferating T cells in CKO cells was significantly reduced (Fig. 2G). The expression levels of T cell activation / proliferation markers IL2RA (CD25), IL-2RB (CD122), CD44, and CD69 were also reduced (Fig. 2H), and could be restored upon re-expression of LARP4 in CKO cells (Fig. 5). Therefore, CKO T cells are less responsive to activation signals and more difficult to activate.

[0063] Furthermore, an LCMV (lymphocytic choriomeningitis virus) infection model was used to investigate the proliferation of antigen-specific T cells in vivo. On day 8 post-infection, compared to control mice, CKO mice showed significantly higher levels of gp66 tetramer-positive CD4+. + The number of T cells was reduced (Figure 2I). To avoid the impact on thymus development, mice expressing the tamoxifen-inducible Cre transgene (ERT2-Cre) were compared with Larp4 mice. fl / fl Mouse hybridization resulted in mice with induced deletion of LARP4 (called iLarp4). - / - (Mouse). From iLarp4 - / - (CD45.2 + ) and ERT2-Cre(Larp4 + / + Mouse, CD45.2 + )Initial CD4 in mice + T cells were transferred to recipient mice (CD45.1). + In this case, the patient underwent four consecutive days of tamoxifen treatment and was subsequently infected with LCMV. (This is related to Larp4.) + / + Compared to mice, on day 8 post-infection, the levels of iLarp4... - / - CD45.2 in mice + CD4 + The expression levels of gp66 tetramer and CD69 and CD44 in T cells were significantly reduced (Fig. 2J). In the LCMV model, LCMV-specific IgM and IgG in CKO mice were only slightly suppressed (Fig. 2K). Finally, the use of CD4-cre also reduced the expression of CD8-... + LARP4 deletion in T cells was investigated, and the effect of LARP4 deficiency on CD8 was examined. + The effect of T cell quiescent state exit, and found to be related to CD4 +T-cell similar phenotype (Figure 6). However, the specific targets of LARP4 and its role in CD8 are unclear. + The role of LARP4 in T cells needs further clarification. Overall, these results suggest that LARP4 may act as a positive regulator of antigen-specific T cell clonal expansion in vivo.

[0064] (2) LARP4 promotes the exit from the resting state in naive T cells by regulating mRNA stability.

[0065] To further investigate the effect of LARP4 on T cell activation, RNA sequencing analysis was performed to identify naïve CD4 from control and LARP4 CKO mice. + Differentially expressed genes among T cells (Figure 7A). Gene set enrichment analysis (GSEA) showed that genes downregulated in the LARP4 CKO background were enriched in energy metabolism, cell cycle, and T cell activation pathways, consistent with the phenotypic characteristics of LARP4 CKO cells (Figure 7B).

[0066] LARP4 is an RNA-binding protein involved in regulating mRNA stability. RIP-seq experiments were then performed to identify naïve CD4. + LARP4-bound mRNAs in T cells. Among the 3310 potential targets identified (Fig. 7C), T cell activation and differentiation pathways ranked in the Top 20 of the GO analysis results, along with RNA splicing and metabolism (Fig. 7D). eCLIP-seq data of LARP4 in K562 and HepG2 cell lines were also used to analyze potential binding preferences. LARP4 binding events were found to be highly enriched at the ends of 3'UTRs (Fig. 8). These results suggest that the interaction between LARP4 and the 3'UTRs of the corresponding mRNAs may contribute to target specificity. As an orthogonal method, genome-wide RNA stability analysis identified LARP4 CKO-initial CD4. +4754 mRNAs with accelerated degradation rates in T cells (Figs. 7E and 7F). Further integration of these three datasets yielded 158 LARP4 functional targets (Fig. 7G), which directly bind to LARP4 and exhibit reduced mRNA stability under LARP4 CKO conditions, ultimately leading to decreased mRNA expression. GO analysis of these 158 genes showed that all of the top 20 enriched pathways were associated with the regulation of immune responses, particularly T cell activation and differentiation (Fig. 7H). Low-throughput validation of several high-confidence targets (Cd3e, Cd2, Cd44, Icos, and Batf) yielded results consistent with genome-wide findings. Furthermore, the size of the amplified products after poly(G / I) tail addition, reverse transcription, and poly(A) PCR using gene-specific primers and tail primers reflected the distribution of poly(A) lengths. For the aforementioned five genes, downregulation of PCR products in CKO samples (compared to controls) was observed by agarose gel electrophoresis, indicating a shortening of the corresponding gene's poly(A) length (Fig. 9). Furthermore, sequence analysis confirmed the overall shortening of the poly(A) tail (Figure 9). Combined with previous observations, these data suggest that LARP4 regulates naïve CD4 by directly binding to and stabilizing its corresponding transcript. + Robustness of T cell exit from resting state and / or activation. Although LARP4 itself is not essential for T cell activation, in the absence of LARP4, the initial CD4+... + T cell activation is reduced, and more cells remain in a resting state.

[0067] (3) The role of LARP4 in helper T cell differentiation

[0068] Inspired by research on T cell stimuli, the next step was to investigate whether LARP4 deficiency affects CD4. + The LARP4 CKO has long-term effects on T cell differentiation. Flow cytometry analysis showed that LARP4 CKO primarily affected naïve CD4. + The baseline levels of T cell differentiation into Th17 and Th1 subsets were observed, but no effect was found on Th2 and Treg subsets (Fig. 10A-D). These observations were further validated by mRNA levels of representative cytokines and transcription factors (Fig. 10A-D). Whole-cell analysis may mask true differences, especially for rare cell subsets; therefore, single-cell RNA sequencing analysis was subsequently performed to address this issue (Fig. 10E, 10F). Consistent with previous results (Fig. 2B), conditional knockout of LARP4 increased naïve CD4 levels. + Size of T cell subsets. Regarding CD4 cell differentiation. +Besides Th1 and Th17 cells, the frequency of Th2 cells was also significantly reduced (Fig. 10G, 10H), likely due to the higher resolution of the single-cell approach. In contrast, the reduction in Treg subsets remained insignificant at the single-cell level (Fig. 10G, 10H), suggesting that LARP4 inhibits CD4+. + The impact on T cell differentiation may be more pronounced on helper (Th) cell subsets. In vitro cell polarization experiments showed that LARP4 deficiency significantly inhibited Th17, Th1, and Th2 differentiation (Fig. 10I-K), leading to decreased levels of IL-17A, IFN-γ, and IL-4 in the culture supernatant, respectively (Fig. 10I-K). Re-expression of LARP4 in CKO cells rescued the differentiation of these subsets (Fig. 10I-K). Then, initial CD4+ labeled with CFSE was used... + Helper T cell polarization experiments were performed on T cells. The results showed that CKO dividing cells (CFSElow) produced fewer cytokines such as IFN-γ, IL-4, or IL-17A (Fig. 11A-C). However, differentiated cells, including Th1, Th2, and Th17 cells, showed similar cytokine production between CKO and control mice in quadrant Q1 of flow cytometry analysis (Fig. 11A-C). These results indicate that LARP4 deficiency leads to reduced overall cytokine production due to impaired cytokine production and slowed cell division. Furthermore, cells were collected on day 5 of polarization and restimulated with the same number of live control or CKO cells using anti-CD3 / anti-CD28. The results showed that cytokine production in control T cells was significantly higher than that in CKO cells (Fig. 11D). These data combined clearly demonstrate the role of LARP4 in the production of naïve CD4 cells. + Effective differentiation of T cells into helper T cell subsets is essential.

[0069] (4) LARP4 deficiency improves autoimmune and allergic diseases in the body.

[0070] Next, we studied LARP4 on CD4. + The in vivo effects of T cell deficiency on several immune diseases. First, a mouse model of autoimmune encephalomyelitis (EAE) was used. This model is well-suited for studying autoimmune inflammatory diseases of the central nervous system and is similar in many ways to human multiple sclerosis (MS), thus it can be used to simulate MS. Injection of myelin oligodendrocyte glycoprotein (MOG) into mice induced monocyte inflammatory infiltration (CD4+). +T cells and macrophages) and demyelination of the white matter surrounding the spinal cord (Fig. 12A). LARP4 CKO significantly slowed disease progression in the corresponding mice (Fig. 12A). Inflammatory cell infiltration and demyelination in the spinal cord were significantly reduced (Fig. 12B). Flow cytometry further confirmed the presence of CD4+ in the spinal cord. + Reduced T cell infiltration (Fig. 12C) was consistent with moderate disease severity in CKO mice. The reduced T cell infiltration was specific to Th17 T cells, while CD4+ IFN in the spinal cord was also reduced. - γ + Th1 cells, CD4 + IL - 4 + Th2 cells and CD4 + FOXP3 + The percentage of Treg cells was similar in CKO and control mice (Fig. 12D and Fig. 13A-C). To confirm that the reduction in disease severity in CKO mice was associated with Th17, CD4 cells isolated from draining lymph nodes were... + T cells, further in vitro with MOG 35-55 Co-culture for an additional 3 days. Enzyme-linked immunosorbent assay (ELISA) showed reduced production of IL-17A and IFN-γ in CKO mouse-derived samples, while IL-4 production was unaffected (Figure 13D). Flow cytometry analysis of MOG-I-Ab tetramers showed that I-Ab MOG in CKO mice was significantly reduced. 35-55 Tetramer-positive CD4 + The CKO mice had fewer T cells than the control mice, indicating reduced T cell activation (Figure 14A). However, I-Ab MOG 35-55 Tetramer-positive CD4 + There was no difference in cytokine production by T cells between CKO and control (Figure 14B). Therefore, the loss of LARP4 may lead to reduced T cell activation, but does not affect the production of cytokines (IFN-γ, IL-17A) by antigen-specific T cells. These results indicate that the loss of LARP4 effectively alleviates EAE.

[0071] Next, a house dust mite (HDM) model was used to simulate Th2-dependent allergic airway disease (Fig. 12E), which was used to simulate asthma. LARP4 CKO and control mice were intraperitoneally injected with HDMs on days 0 and 14, followed by intratracheal (it) administration of HDMs on days 21 and 24, respectively. Compared with PBS, HDM exposure resulted in an increase in total cell count and eosinophil count in bronchoalveolar lavage fluid (BAL). However, in LARP4 CKO mice, cell infiltration was moderately suppressed (Fig. 12F). LARP4 CKO mice also showed reduced lung pathology, namely less mucus and inflammation (Figs. 12G, 12H), highlighting that LARP4 deficiency alleviates allergic airway disease. All these data suggest that attenuated CD4 + T cell differentiation may be limited to those heavily dependent on naïve CD4. + Diseases caused by abnormal activation and differentiation of T cells into specific helper T cell subsets.

[0072] Example 2: Design of LARP4 inhibitory peptide LIPEP

[0073] (1) Interaction between LARP4 and PABP

[0074] Studies have found that LARP4-mediated mRNA stability requires the interaction of the LARP4 PAM2 motif with the MLLE domain of PABP or PABPC (see upper part of Figure 15A for a schematic diagram). LARP4-related mRNA stabilization activity requires the interaction of its PAM2 motif with the MLLE domain of PABP, and this interaction can facilitate the initial CD4+ molecule formation. + T cells prevent deadenylation.

[0075] The amino acid sequence of the PAM2 motif of LARP4 (La ribonucleoprotein 4; Gene ID 207214) is: TGLNPNAKVWQE (SEQ ID NO.2, the PAM2 motif of human LARP4 is completely identical to that of mouse LARP4).

[0076] The amino acid sequence of MLLE in PABP (poly(A)binding protein) or PABPC (poly(A)binding protein, cytoplasmic1; Gene ID 18458) is: QEQKQMLGERLFPLIQAMHPSLAGKITGMLLEI (PABP) 556-588(SEQ ID NO.3, the mouse and human sequences differ by only one amino acid ST). Poly(A)binding proteins (PABPs) are a class of highly conserved RNA-binding proteins, widely found in eukaryotes, capable of specifically recognizing and binding to poly(A) nucleotide sequences. The PABP family is mainly divided into two types based on their location within the cell: nuclear poly(A)binding protein (PABPN) and cytoplasmic poly(A)binding protein (PABPC).

[0077] (2) Design LIPEP based on MLLE domain

[0078] Further research by the inventors revealed that blocking the interaction between LARP4 and PABP achieves effects similar to LARP4 deficiency in vitro and in vivo (see Figure 15A, lower part for schematic diagram). Specifically, blocking this interaction leads to reduced T cell activation, thereby alleviating autoimmune and allergic diseases. The inventors demonstrated this effect using a LARP4 inhibitory peptide (LIPEP) that blocks the interaction between LARP4 and PABP. This project aims to develop an MLLE domain-mimicking peptide called LARP4 inhibitory peptide (LIPEP) to block the binding of LARP4 to PABP. LIPEP (a competitive peptide inhibitor of LARP4) was independently designed by the inventors, and its sequence is as follows: (SEQ ID NO.1, double underlines indicate mutation sites that increase hydrophilicity, and wavy lines indicate mutation sites that increase affinity; Palm indicates N-terminal palmitic acid modification, and NH2 indicates C-terminal amidation modification). The LARP4 inhibitory peptide (LIPEP) is designed based on the MLLE domain of PABP, and several mutations are introduced at the binding interface (Figure 15B). The specific mutation sites are: GLU 564 Mutate into LYS, LYS 580 The mutation to GLU, these two key sites are crucial for the affinity between LIPEP and PARP4; in addition, LEU 562 ,LEU 569 ILE 588Mutations to THR, SER, and THR, respectively, further improved the hydrophilicity of LIPEP. In addition, subsequent experimental studies involved further modifications to LIPEP. Based on the LIPEP amino acid sequence, conventional peptide modifications were performed at the N and C ends to mimic the natural state of the peptide. Specifically, palmitic acid was added to the N-terminus, and the C-terminal carboxyl group was amidated. Chemically synthesized peptides typically possess free amino groups (N-terminus) and free carboxyl groups (C-terminus). These terminal modifications generate mimics that more closely resemble natural proteins, thereby enhancing peptide stability. The conventional method for modifying the free carboxyl group (C-terminus) is amidation, where the free carboxyl group is reacted to form an amide group. The conventional method for modifying the free amino group (N-terminus) is palmitic acid addition, where palmitic acid is linked to the N-terminal free amino group, thus blocking the free amino group. The LIPEP peptide sequence synthesis and the N- and C-terminal modifications were all obtained by a third-party company using conventional methods. The biological function verification in subsequent embodiments all used peptides prepared by third-party companies. Computer-aided peptide function studies primarily focused on the peptide sequence.

[0079] Regarding the mutation sites of LIPEP, the inventors discovered that the key positions for the interaction between the PAM2 motif of LARP4 and the MLLE domain are P9 and P12. At the P9 position, the amino acid in the MLLE domain of PABP is the basic amino acid K (LYS). 580 At the P12 position, the amino acid in the MLLE domain of PABP is the acidic amino acid E (Glu). 564 Point mutation studies were conducted targeting the P12 site, specifically targeting GLU... 564 The mutation resulted in the formation of basic amino acids such as HIS, ARG, and LYS; however, only GLU was found to be mutated. 564 The peptide only acquires its biological activity after being mutated to LYS. Point mutation studies were conducted targeting the P9 site, specifically modifying LYS... 580 Mutations to acidic amino acids such as ASP and GLU were found, however, only LYS was found to be mutated. 580 The peptide only acquires biological efficacy after being mutated to GLU.

[0080] In addition, the inventors also discovered some sites where the MLLE domain does not interact with the PAM2 motif. They selected hydrophobic amino acids from these unrelated sites and mutated them to hydrophilic amino acids in the precursor without altering the peptide's secondary structure, thus completing the hydrophilic modification of the peptide. Based on these two key site mutations, they further enhanced the peptide's ability to block the interaction between the LARP4 PAM2 motif and the MLLE domain.

[0081] (3) Evaluation of the effect of LIPEP in blocking the interaction between LARP4 and PABP

[0082] Based on the crystal structure (3PKN) of the complex formed by the MLLE domain of PABP and the PAM2 motif of LARP4, interaction models of LIPEP or MLLE with all collected PAM2 motifs were generated. Stable configurations of each complex were collected after dynamic calculations. The 20 complexes of PAM2 motifs with LIPEP can be divided into two main groups based on the configuration of the key position P10 in the PAM2 motif: Group A, where the aromatic residues at the P10 position of the PAM2 motif maintain a similar configuration to those in the MLLE complex (Fig. 15C); and Group B, where the aromatic residues at the P10 position of the PAM2 motif protrude from the original binding groove (Fig. 15D). According to the interaction model of PAM2 motifs with the MLLE domain, the binding configurations in Group B may represent non-specific binding. LIPEP belongs to Group A, and LIPEP shows the lowest interaction energy with the PAM2 motif of LARP4. Comparing the interactions of LIPEP or MLLE with the PAM2 motif of LAPR4, LIPEP exhibits a more hydrophobic interaction surface than MLLE (Figs. 15E and 15F).

[0083] The most significant difference in the interaction between the LIPEP-LARP4 and MLLE-LARP4 complexes is at position P564. The Glu-to-Lys mutation at this corresponding position in LIPEP reduces the two unfavorable interactions observed in the MLLE-LARP4 complex and generates a strong interaction (attraction) between this residue in LIPEP and Glu in the PAM2 motif of LARP4 (Figures 15G and 15H). The LIPEP provided by this protocol primarily competitively blocks the endogenous LARP4-PABP interaction without affecting the binding of MLLE to other PAM2-containing proteins (such as PAIP2 and PAN3) (Figure 15I). In the table of Figure 15I, the LARP4-LIPEP interaction energy is -226.6 kcal / mol, lower than the LARP4-PABP (MLLE domain) interaction energy (-200.3 kcal / mol), indicating that LIPEP effectively blocks the binding of LARP4 and the MLLE domain, thereby inhibiting downstream signaling pathways. The interaction energies between LIPEP and other proteins containing the PAM2 motif in the table of Figure 15I are all higher than the interaction energies between these proteins and the MLLE domain, indicating that LIPEP does not competitively inhibit the binding of other proteins containing the PAM2 motif to the MLLE domain. Furthermore, the interaction energies between LIPEP and other proteins containing the PAM2 motif in the table of Figure 15I are all higher than the interaction energies between LARP4 and LIPEP, further demonstrating the strong specificity of the binding between LARP4 and LIPEP. These results demonstrate that the LIPEP designed in this protocol, through a specific amino acid point mutation in the MLLE domain, effectively blocks the binding of LARP4 to downstream molecules (PABP containing the MLLE domain), thereby blocking the corresponding LARP4-mediated signaling pathway and achieving control of the T cell resting state. Moreover, the specific amino acid point mutation design also ensures that LIPEP does not affect the functional pathways of other proteins containing the PAM2 motif. Therefore, it can be seen that the design of LIPEP in this scheme is non-obvious compared to the prior art, and achieves unexpected technical effects.

[0084] Since not all PAM2-containing proteins have readily available commercially available antibodies for immunoprecipitation (IP) studies, this protocol recombinantly expressed each PAM2-containing protein (with an attached tag) for use in immunoprecipitation studies. Fourteen different plasmids were constructed, each expressing a PAM2-containing protein with a different tag. LARP4 was tagged with an N-terminal HA tag, while the other 13 PAM2-containing proteins were tagged with Flag, Myc, or His epitopes, respectively. The LARP4-expressing plasmid was co-transfected with two or three other PAM2-containing protein plasmids (up to four, with four different protein tags) simultaneously for LIPEP treatment in equimolar proportions. The experimental design allowed for side-by-side comparison of multiple PAM2-containing proteins to minimize transfection variability. Immunoprecipitation (IP) was then performed on the various PAM2-containing proteins, followed by semi-quantitative detection of bound LIPEP by Western blotting (Figure 15j) and fluorescence detection (Figure 15k). The relative amount of LIPEP was further normalized by the IP efficiency of each labeled protein to correct for differences introduced in the IP step (Figures 15J and 15K). The results showed that LARP4 was the major target of LIPEP compared to other PAM2-containing proteins.

[0085] (4) Pharmacokinetic / pharmacodynamic studies and mechanism of action studies of LIPEP

[0086] Detailed pharmacokinetic / pharmacodynamic data for LIPEP treatment in mice are shown in Figures 15L and 15M. Results in mice indicated that the half-life of LIPEP in mouse plasma was 5.3 hours (Figures 15L and 15M). Notably, the safety of LIPEP treatment was also assessed through pathological and serological investigations, with virtually no obvious toxic side effects detected (Figure 16).

[0087] Flow cytometry and confocal fluorescence microscopy analysis showed that LIPEP was able to enter the in vitro naïve CD4+ cells. + T cells (Figs. 18A and 18B). In Fig. 18A, confocal fluorescence microscopy was used to detect the entry of FITC-labeled LIPEP at a concentration of 4 μM into naïve CD4 cells. + T cells were analyzed using DAPI (4,6-diamino-2-phenylindole; D9542; Sigma-Aldrich) to label the cell nuclei. In Figure 18B, flow cytometry was used to detect the entry of FITC-labeled LIPEP into the initial CD4+ cells at different concentrations. +In the case of T cells, control cells were treated with DMSO. LIPEP had no effect on cell death or apoptosis (Fig. 17), and co-immunoprecipitation experiments revealed that LIPEP could block the binding of LARP4 to PABPC1 in a dose-dependent manner (Fig. 18C). In 18C, LIPEP treatment inhibited the interaction between LARP4 and PABPC1. HeLa cells were transfected with pcDNA3.1-PABPC1 Flag and pcDNA3.1-LARP4 HA vectors, and then treated with different concentrations of LIPEP for 24 hours. Whole-cell lysates of HeLa cells were immunoprecipitated (IP) with anti-HA antibody or control mouse IgG, followed by Western blotting (IB) with anti-Flag or anti-HA HRP antibody. The internal control protein was also IB-ed with anti-Flag or anti-HA HRP antibody, and GAPDH was used as an internal control. As expected, the initial CD4... + The interaction between LARP4 and its corresponding mRNAs (e.g., Cd2 and Cd3e) in T cells is partially blocked by LIPEP (Fig. 18D), resulting in mRNA instability (Fig. 18E) and reduced expression levels (Fig. 18F).

[0088] Example 3: LIPEP attenuated T cell activation / differentiation in vitro.

[0089] The effects of LIPEP treatment on in vitro initial CD4 levels were investigated. + Effects on T cells. First, LIPEP treatment of naïve CD4 cells. + T cells increased the expression of multiple resting regulatory factors (Klf2, Klf6, and Foxp1) (Fig. 19A) and led to an increase in the proportion of cells in the G0 phase (Fig. 19B). Furthermore, LIPEP was added to naïve CD4 during T cell activation. + Cell proliferation was assessed by measuring Ki-67 incorporation at a fixed time point (24 hours) after the addition of LIPEP in the culture medium for T cells. When LIPEP was added at the onset of anti-CD3 / CD28 stimulation, the percentage of cells entering the G1 or G2-SM phase decreased significantly in a concentration-dependent manner (Fig. 19C). However, when LIPEP was added 24 hours after TCR stimulation, the effect was much milder, and there was almost no effect when introduced at the 48-hour time point (Fig. 19C). This time dependence is consistent with the expression dynamics of LARP4 during T cell activation, suggesting that LARP4 plays a role in the early cell cycle entry phase (Fig. 20).

[0090] Next, we investigated whether LIPEP treatment affected the differentiation of Th1, Th2, and Th17 cells in vitro. In different LIPEP treatment groups, after LARP4 overexpression, we studied the effects of LIPEP on PAM2-containing proteins, mRNAs, and cytokines on CD4+. + The effects of expression in T cells, including these proteins, mRNAs, and cytokines associated with autoimmune and allergy markers, were investigated. Results showed that, except for LARP4, the relative levels of mRNAs encoding other PAM2-containing proteins did not change significantly (Figure 21A). Further validation of LARP4 and LARP4B protein levels yielded results consistent with mRNA levels (Figure 21B). Furthermore, overexpression of LARP4 in LIPEP-treated cells was found to have a rescue effect on the expression levels of mRNAs and cytokines associated with autoimmune and allergy markers such as IL-17A and IL-4 (Figure 21C). Combined with the results shown in Figure 15, it can be concluded that LARP4 may be the primary therapeutic target of LIPEP. Similar to the LARP4 CKO condition, LIPEP was applied at the start of the polarization program, significantly blocking CD4. + Differentiation of helper T cells. When LIPEP was added at the 24-hour time point, the attenuation effect was present but not very significant, while when LIPEP was added at the 48-hour time point, this effect almost disappeared (Figs. 19D-19F). These findings indicate that LIPEP can attenuate the differentiation of all three T helper cell lineages in vitro. Based on evidence of high homology between the amino acid sequence of LIPEP and the MLLE of human PABP, its role in human T cells was also investigated. As expected, LIPEP was able to effectively enter human CD4+. + T cells primarily block the binding of LARP4 to its target mRNA, thereby reducing the stability and expression level of the target mRNA (Figure 22).

[0091] Finally, an adoptive T-cell transfer model was used to evaluate the in vivo effects of LIPEP treatment. Initial CD4 cells were purified from SMARTA mice (CD45.1). + T cells were collected and treated with either LIPEP or mannitol. Eight hours after treatment, both cell populations were transferred into CD45.2 mice. The mice were then infected with LCMV and sacrificed eight days post-infection. Results showed that, compared to the control group, mice treated with LIPEP showed significantly higher initial CD45.2 cell counts. + T cells in mouse spleen, CD45.1 + Vα2 + Antigen-specific T cells (CD45.1) + Vα2 + CD4 + CD69 +And CD45.1 + Vα2 + CD4 + CD44 + The absolute number of T cells was significantly reduced (Figure 19G), indicating that treatment with LIPEP on naïve CD4 cells... + T cells, and the use of the above cells to treat mice, can inhibit the clonal expansion of antigen-specific T cells in vivo.

[0092] Example 4: The effect of LIPEP depends on the presence of LARP4

[0093] To exclude LIPEP from the initial CD4 + The effect on T cells may be due to the possibility of non-specific or indirect interactions, as seen in the initial CD4 cell treatment from LARP4 CKO mice using LIPEP. + T cells in these mice lacked the LIPEP target. In this case, the expression level and stability of Cd3e mRNA were unaffected by LIPEP (Figs. 23A and 23B). Similar results were observed for the other two resting regulators, Klf2 and Klf6 (Figs. 23C and 23D). More importantly, under in vitro differentiation conditions, LIPEP treatment did not further attenuate differentiation into all corresponding CD4 cells. + The process of helper T cell subsets (Figure 24). Furthermore, EAE and HDM models were induced in LARP4 CKO mice, followed by treatment with LIPEP. Results showed that LIPEP treatment in LARP4 CKO mice did not contribute to disease improvement in the EAE and HDM models. Since there was no additive effect between LARP4 CKO and LIPEP, this indicates that the effects of LIPEP in vitro and in vivo are LARP4-dependent.

[0094] Example 5: LIPEP simulated the LARP4 CKO phenotype in multiple mouse disease models.

[0095] This study investigated the potential of LIPEP for the prevention and treatment of disease in vivo. In an EAE mouse model, the prevention group received subcutaneous (sc) injections of LIPEP on days 1, 4, 7, and 10 (Fig. 25A), while the treatment group received injections on days 12, 15, and 18, or days 15, 18, and 21 (Fig. 26A). Clinical scores showed that the disease severity was alleviated in these LIPEP-treated groups (Figs. 25B and 26B), and inflammatory cell infiltration and demyelination in the spinal cord were significantly reduced (Figs. 25C and 26C). CD4 isolated from the spinal cord of diseased mice was analyzed by flow cytometry. +T cells further confirmed the reduction in cell infiltration (Figs. 25D and 26D). The proportion of Th17 cells in the spinal cord of LIPEP-treated mice was significantly reduced (Figs. 25E and 26E), while the percentages of Th1, Th2, and Treg cells in the spinal cord were similar between the LIPEP and control groups (Figs. 25F-25H and 26F-26H). Similar results were observed in T cells isolated from lymph nodes (Fig. 25I). These results demonstrate the potential of LIPEP for the prevention and treatment of autoimmune diseases.

[0096] In a Th2-dependent allergic airway disease model, mice were first sensitized with HDM, followed by subcutaneous (sc) injections of LIPEP or a carrier solution on days 1, 4, and 7 in the prevention group (Fig. 27A) and on days 11, 14, and 17 in the treatment group (Fig. 28A). Following re-exposure to HDM (days 21 and 24), mice were sacrificed on day 25 to assess disease severity. Mice receiving LIPEP injections showed significant remission, evidenced by a modest reduction in total cell count and eosinophil count in bronchoalveolar lavage fluid (BAL) (Figs. 27B and 28B) and moderate pulmonary pathological changes (Figs. 27C, 27D and 28C, 28D), highlighting the potential of LIPEP in the prevention and treatment of allergic diseases.

[0097] In summary, LARP4 deficiency can lead to the exit of naïve T cells from the resting state (Figure 29). Regarding LARP4-mediated naïve CD4... + The regulatory mechanism of T cell exit from the resting state: LARP4 can induce naive T cells to exit the resting state by promoting the prolongation and stabilization of mRNA PAT of factors related to T cell activation (such as Cd3e and Cd2), and by protecting naive CD4+. + T cells are protected from deadenosylation, thereby reducing initial CD4 levels. + The threshold for T cell activation. Treatment with LIPEP can block the binding of LARP4 to other molecules, thereby causing naive T cells to exit the resting state.

[0098] Comparative Example 1: Comparison of the bioactivities of different peptides

[0099] Biological function studies were conducted on the peptides listed in Table 1. Mouse CD4 cells were treated with the peptides listed in Table 1 at concentrations of 0 μM (control), 4 μM, and 6 μM. +T cells were thawed for 12 hours, and then the RNA expression levels of genes such as Rorc, Tbx21, Gata3, IL17a, Ifng, and IL4, as well as the protein expression levels of genes IL17a, IFNγ, and IL4, were tested. This comparative example demonstrates the process of detecting the biological activity of candidate proteins through phenotypic inhibition; the experimental results are shown in Figures 30-32. The results show that replacing the E in Pep-KE with the D in Pep-KD, and replacing the K in Pep-KE with the R in Pep-RD, results in the peptide losing its biological function. The above experimental studies demonstrate that point mutations in proteins generally lead to loss of protein function; only by mutating amino acids at specific sites to specific amino acids can the mutated peptide inhibit the interaction between LARP4 and PABP. The determination of the mutation sites and mutation methods in this protocol has yielded unexpected technical effects in regulating T cell quiescent exit, treating autoimmune diseases, and allergic diseases.

[0100] Table 1: Candidate peptide sequence information

[0101] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A LARP4 inhibitor, LIPEP, characterized in that: It is used to block the interaction between LARP4 and PABP.

2. The LARP4 inhibitor LIPEP according to claim 1, characterized in that: LARP4 interacts with the MLLE domain of PABP through the PAM2 motif.

3. The LARP4 inhibitor LIPEP according to claim 2, characterized in that: The amino acid sequence of the PAM2 motif of LARP4 is shown in SEQ ID NO.2; the amino acid sequence of the MLLE domain of PABP is shown in SEQ ID NO.

3.

4. The LARP4 inhibitor LIPEP according to claim 3, characterized in that: The amino acid sequence of LIPEP is shown in SEQ ID NO.

1.

5. The use of the LARP4 inhibitor LIPEP according to any one of claims 1-4 in the preparation of a medicament for treating or preventing diseases caused by abnormal T cell activation or T cell dysfunction.

6. The use of the LARP4 inhibitor LIPEP according to claim 5 in the preparation of a medicament for treating or preventing diseases caused by abnormal T cell activation or T cell dysfunction, characterized in that: Diseases caused by abnormal T cell activation or T cell dysfunction include autoimmune diseases and allergic diseases.

7. The use of the LARP4 inhibitor LIPEP according to claim 6 in the preparation of a medicament for treating or preventing diseases caused by abnormal T cell activation or T cell dysfunction, characterized in that: The autoimmune diseases mentioned include multiple sclerosis.

8. The use of the LARP4 inhibitor LIPEP according to claim 6 in the preparation of a medicament for treating or preventing diseases caused by abnormal T cell activation or T cell dysfunction, characterized in that: The allergic diseases mentioned include asthma.

9. Application of LARP4 in screening or designing drugs that regulate the exit of naïve T cells from the resting state.

10. The use of LARP4 according to claim 9 in screening or designing drugs that regulate the exit of naive T cells from the resting state, characterized in that; Drugs that regulate the exit of nascent T cells from the resting state include: competitive peptide inhibitors that inhibit the interaction between the PAM2 motif of LARP4 and the MLLE domain of PABP, or systems that inhibit the expression of the LARP4 gene or protein.

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