KR102543157B1 - Toll-like receptor 1/2 and/or 4 Activating Peptides and Uses Thereof - Google Patents

Abstract

Acidovorax avenae is a flagellar bacterium that is pathogenic to a variety of plant crops and is also found in patients with blood cancer, fever or sepsis. However, the exact mechanism of infection in humans is unknown. We postulated that the human immune system responds to purified flagellin ('FLA-AA') isolated from the bacterium. In the present invention, human macrophages, fibroblasts, and TLR5-overexpressing cell lines were treated with FLA-AA to confirm activation of proinflammatory signaling pathways through TLR5 and NLRC4 at the molecular level. In order to find effective regions in FLA-AA, four peptides ('AF1 to AF4') were designed from the α-helix of the N and C terminals, and their efficacy was analyzed in several cell types. Peptides AF1 and AF2 showed agonistic activity through TLR1/2 and TLR4, but not through TLR5. This suggests cross-reactivity of flagellin fragments with members of the same family of toll-like receptors. Short flagellin fragments have potential as therapeutic agonists or adjuvants in cancer immunotherapy.

Description

Toll-like receptor 1/2 and/or 4 Activating Peptides and Uses Thereof

The present invention relates to peptides that activate TLR (Toll-like receptor) 1/2 and/or TLR4 signaling pathways, and more specifically, to activate TLR1/2 and/or TLR4 signaling pathways to secrete pro-inflammatory cytokines And a peptide inducing activation of NF-κB and MAPKs, a TLR agonist containing the peptide, an adjuvant, a composition for preventing or treating cancer, and a composition for preventing or treating TLR1/2 and/or TLR4 related diseases. .

Acidovorax avenae is an oxidase-positive, non-lactose degrading, aerobic, rod-shaped, achromatic, gram-negative bacterium that is economically important, such as rice, sorghum, corn, millet, sugarcane, and oats. Pathogenic to a variety of crops (Song, W. et al., Journal of Phytopathology 2004, 152 , 667-676). It causes a deadly rice disease called bacterial brown stripe in parts of Africa, Asia, Europe and North America. Although this species causes great damage to plants, little is known about opportunistic infections in humans. The presence of A. avenae was confirmed in hematological cancer and fever patients by PCR amplification, and it was confirmed in sepsis patients by whole-cell long-chain-fatty-acid analysis. Another species , Acidovorax oryzae have also been reported to cause bacteremia in humans with catheters. Acidovorax spp. were initially classified as Pseudomonas spp., and their role as animal or human pathogens was not investigated in detail (Willems, A. et al., International Journal of Systematic and Evolutionary Microbiology 1990, 40 , 384-398).

Pathogens have specific signaling molecules called "pathogen-associated molecular patterns (PAMPs)," which act on host pattern recognition receptors (such as Toll-like receptors (TLRs)). PRR) and triggers the human innate immune system (Medzhitov, R. et al., Nature 1997, 388 , 394) TLRs are specific with the help of leucine-rich repeats in their extracellular domain. It is a type I transmembrane protein with extracellular, transmembrane and intracellular domains that recognize PAMPs These TLRs are located on the cell surface or in the membrane of endosomes or lysosomes and interact with their respective ligands (Uematsu, S. et al. al., In Toll-like receptors ( TLRs ) and innate immunity , Springer: 2008; pp. 1-20) TLRs are activated by various bacterial products: peptidoglycan, lipoarabinomanan and bacteria Lipoproteins interact with TLR2, lipopolysaccharide (LPS) is primarily involved in TLR4, bacterial flagellin is recognized by TLR5 Endosomal TLRs recognize a diverse set of ligands Double-stranded RNA is involved in TLR3 and single-stranded RNA is detected by TLR7 and TLR8, which interact with viral and bacterial CpG DNA motifs and the malaria pigment hemozoin. /interleukin-1 receptor (TIR) domain interacts with cytoplasmic adapter molecules containing the TIR domain, eventually NF-κB (nuclear factor kappa light-chain-enhancer of activated B cells), AP-1 (activating protein) 1) and IRFs (interferon-regulatory factors) are activated (Gay, NJ et al., Nature Reviews Immunology 2014, 14 , 546). These transcription factors regulate the expression of several cytokines to prepare the host for invaders. Nevertheless, inappropriate activation of TLRs can lead to various health problems such as cancer, rheumatoid arthritis and sepsis.

Toll-like receptors (TLRs) are major membrane-bound pattern recognition receptors (PRRs) located on cell membranes and endosomal membranes, recognizing molecular patterns associated with pathogens or damage, and expressing tumor necrosis factors. Down, which is completed by the expression of pro-inflammatory cytokines such as α (tumor necrosis factor α; TNF-α), interleukin 1 (IL-1), IL-6, IL-10, and type I interferons (IFN-α/β) It causes a complex cascade of stream signaling. These molecules induce leukocytes to migrate to the site of infection, activate antigen-presenting cells, and then initiate T cell differentiation for a sustained adaptive immune response.

Among the cell surface TLRs, TLR2 heterodimerizes with TLR1 or TLR6 to recognize a broad spectrum of pathogenic components, such as triacylated lipopeptides (Pam3CSK4), diacylated lipopeptides (Pam2CSK4), lipoteichoic acids, zymosan, bacterial and fungal lipids, acylated sugars, and proteins. form a unique TLR2 has been shown to associate with TLR10 for the recognition of triacylated lipopeptides, but the clear mechanism or cytokines released by this pathway are unknown (Guan Y, et al. (2010) Journal of immunology 184(9):5094- 5103). Activated TLR2 recruits the myeloid differentiation primary response 88 (MyD88) protein and is assisted by the Toll/interleukin-1 receptor (TIR) homology domain-containing adapter protein (TIRAP) through TIR domain interactions. MyD88 recruits interleukin-receptor-associated kinase 4 (IRAK4), which activates IRAK1 through phosphorylation. The actual stoichiometry of the complex assembly of TLR-MyD88-IRAK4-IRAK1/2 termed the ‘Myddosome’ is not clear. Several adapters and kinases are involved in translocating NF-κB to the nucleus through numerous phosphorylation and ubiquitination for the expression of pro-inflammatory cytokines and chemokines (Kawasaki T & Kawai T (2014) Frontiers in immunology 5:461).

TLRs are attractive targets for vaccine adjuvants because of their ability to activate both the innate and adaptive immune systems. Effective and non-toxic TLR agonists meet essential safety criteria for use with prophylactic vaccines, which have generated great interest in medicine (Casella CR & Mitchell TC (2008) Cellular and molecular life sciences: CMLS 65(20):3231-3240). A number of TLR agonists are currently in various stages of clinical investigation. For example, there is a preclinical report that TLR2 agonists may be the most effective adjuvants for treating HIV, HBV and HPV infections (Borsutzky S, et al. (2006) Vaccine 24(12):2049-2056). Lipopeptide mimetic agonists of TLR2 show strong adjuvant effects with various antigens (Willems MM, et al. (2014) Journal of medicinal chemistry 57(15):6873-6878). Attenuated TLR1/2 signaling is associated with an increase in infection-related morbidity and mortality with aging. The occurrence of these diseases can be prevented by applying a TLR1/2 agonist as monotherapy or combined therapy (Shechter R, et al. (2013) Scientific reports 3:1254), and according to a recent report, TLR2 signaling affects oligodendrocytes. It has been shown to play a central role in preventing ischemic stroke in the white matter, which is characterized by oligodendrocyte (OL) death and demyelination.

TLR2 agonists have shown promising results in reducing lung tumors, bladder cancer, breast cancer, and pancreatic ulcers (Zhang Y, et al., (2011) Journal of immunology 186(4):1963-1969, etc.). Apart from anticancer drugs, TLR1/2 agonists have been shown to be effective in treating various infectious diseases such as chronic and acute influenza, asthma and age-related obesity (Tan AC, et al. (2012) Molecular pharmaceutics 9(9): 2710-2718). However, despite this potential clinical efficacy, high-throughput screening for the discovery of short peptides capable of stimulating TLR2 has not been reported to date except for a few small molecules (Cheng K, et al. (2015) Science advances 1(3)).

Accordingly, the present inventors made diligent efforts to develop a pure peptide-based TLR1/2 or TLR4 agonist, and as a result, a peptide derived from flagellin protein of plant pathogenic bacteria ('AF (A = Acidovorax) avenae ; F = flagellin)') effectively induces an innate immune response through TLR1/2 and/or TLR4 independently of TLR5, so it was confirmed that there is potential for application as an adjuvant or cancer treatment, and the present invention was completed. .

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms prior art known to those skilled in the art to which the present invention belongs. may not be

An object of the present invention is to provide a peptide activating the TLR1/2 and/or TLR4 signaling pathway and a TLR agonist comprising the peptide.

Another object of the present invention is to provide an adjuvant containing the peptide.

Another object of the present invention is to provide a composition for preventing or treating cancer or TLR1/2 and/or TLR4-related diseases comprising the peptide.

Another object of the present invention is to provide a method for preventing or treating cancer or TLR1/2 and/or TLR4 related diseases, which includes administering the peptide.

Another object of the present invention is to provide the use of the peptide for preventing or treating cancer or TLR1/2 and/or TLR4 related diseases.

Another object of the present invention is to provide the use of the peptide for the preparation of a drug for preventing or treating cancer or TLR1/2 and/or TLR4 related diseases.

In order to achieve the above object, the present invention provides a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention also provides an adjuvant containing the peptide.

The present invention also provides a composition for preventing or treating cancer or TLR1/2 and/or TLR4-related diseases comprising the peptide.

The present invention also provides a method for preventing or treating cancer or a TLR1/2 and/or TLR4 related disease comprising administering the peptide.

The present invention also provides the use of the peptide for preventing or treating cancer or TLR1/2 and/or TLR4 related diseases.

The present invention also provides the use of the peptide for the preparation of a drug for preventing or treating cancer or TLR1/2 and/or TLR4 related diseases.

In the present invention, it was confirmed that flagellin (FLA-AA) purified from A. avenae N1141 can trigger the human innate immune system by interacting with TLR5 and NLRC4. While the α-helical peptide fragments of flagellin cannot activate TLR5, the peptide fragments AF1 and AF2 according to the present invention can induce an innate immune response through TLR1/2 and TLR4, thus providing therapeutic value in cancer immunotherapy. It has potential as an agonist or adjuvant.

Figure 1: A diagram showing the alignment of flagellin sequences derived from FLA-AA and FLA-ST. The protein sequences of flagellins derived from FLA-AA and FLA-ST were compared using "EMBOSS water", a pairwise local sequence alignment tool of EMBL-EBI, to evaluate their similarity.
Figure 2: FLA-AA activates immune signaling in human macrophages and fibroblasts. (FIG. 2A, D) The viability of THP-1 macrophages (a) and HDFs (b) was determined by MTT assay after treatment of the cells with FLA-AA or FLA-ST at the indicated concentrations for 24 hours. (FIG. 2b, c, e, f) THP-1 macrophages (b, c) and fibroblasts (e, f) were treated with the indicated concentrations of FLA-AA or FLA-ST for 24 h. Culture supernatants were collected and processed for quantification of cytokines including IL-8 (b, e), TNF-α (c) and IL-6 (f) using respective ELISA kits. (FIG. 2G) After treatment of THP-1 cells with FLA-AA (200 ng/ml) for the indicated periods, Western blotting was performed to analyze NF-κB and MAPK activation. β-Actin was used as an endogenous control. (FIG. 2H) PMA-differentiated THP-1 cells were primed with LPS for 4 hours and transfected with the indicated concentrations of FLA-AA or FLA-ST along with control for 2 hours. Culture supernatants were assayed for secretion of IL-1β using a related ELISA kit. All experiments were independently performed 4 times, and the mean ± SEM difference between the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05). FLA-AA: flagellin from A. avenae ; FLA-ST: flagellin from S. Typhimurium; IL-6: interleukin 6; IL-8: interleukin 8; LPS: lipopolysaccharide; MAPKs: mitogen-activated protein kinases; NF-κB: nuclear factor “kappa-light-chain-enhancer” of activated B cells; PMA: phorbol 12-myristate 13-acetate; TNF-α: tumor necrosis factor α.
Figure 3: FLA-AA mediated signaling is specific to TLR5. (Fig. 3a, b, c, d) THP-1 cells (10 ⁵ /well) (a, b) and HDF (10 ⁴ /well) (c, d) were treated with various concentrations (1.5, 3 or 6 μM) of TH1020. was pretreated for 1 hour and post-treated with FLA-ST or FLA-AA at the indicated concentrations. 24 hours after post-treatment, the culture supernatant of the treated THP-1 cells was analyzed to calculate the levels of human IL-8 (hIL-8) (a) and hTNF-α (b), and the culture supernatant of HDF was Assayed for hIL-8 (c) and hIL-6 (d) by ELISA. (FIG. 3e) The viability of 293/hTLR5 cells (3×10 ⁴ /well) was determined by MTT assay after treatment with the indicated concentrations of FLA-AA or FLA-ST for 24 hours. (FIG. 3F) The degree of hIL-8 secretion by 293/hTLR5 cells (3×10 ⁴ /well) was determined by ELISA after treatment with FLA-AA or FLA-ST at the indicated concentrations for 24 hours. The experiment was independently performed 4 times, and the mean ± SEM difference between the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05).
Figure 4: Immunofluorescence analysis shows the specificity of FLA-AA for TLR5. (FIG. 4a, b) THP-1 macrophages (a) and HDFs (b) were grown overnight in 24-well plates. Cells were then pretreated for 1 hour with the indicated concentrations of TH1020. Post-processing was based on FLA-AA or FLA-ST, and immunofluorescence analysis was performed using specific anti-p-p65 and anti-β-actin primary antibodies and Alexa Fluor-conjugated secondary antibodies. Red represents the phosphorylated subunit (p-p65) of NF-κB, green represents β-actin, and blue represents the nucleus of Hoechst 33258 dye. Images were captured at ×40 magnification using an inverted microscope (Olympus IX53; Olympus Corporation, Tokyo, Japan). Scale bar: 50 μm.
Figure 5: A diagram showing AF peptides derived from FLA-AA. The full-length amino acid sequence of FLA-AA is shown, and the amino acid residues constituting the AF peptide are shown in different colors and/or underlined.
Figure 6: A diagram showing toxicity evaluation of AF peptides. THP-1 cells (10 ⁵ /well) (a) and HDF (10 ⁴ /well) (b) were treated with the indicated concentrations of peptides and FLA-ST for 24 hours, and then MTT assay was performed to calculate cell viability. . All experiments were independently performed 4 times, and the mean ± SEM difference between the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05). FLA-ST: flagellin from S. Typhimurium .
Figure 7: Screening and identification of functional peptides. (FIG. 7a, b) PMA-differentiated THP-1 cells were treated with various concentrations of peptides and FLA-ST for 24 hours. Levels of hIL-8 (a) and hTNF-α (b) secretion were determined by ELISA. (FIG. 7c,d) HDFs were treated with various concentrations of peptide and FLA-ST, and hIL-8 (c) and hTNF-α (d) secretion levels were determined after 24 h by ELISA. (FIG. 7E) After treatment of HDFs with AF1 (5 μM) for the indicated period, Western blot was performed to analyze the activation of NF-κB and MAPK. β-actin was used as an endogenous control. All experiments were independently performed 4 times, and the mean ± SEM difference between the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05). FLA-ST: flagellin from S. Typhimurium; hIL-8: human interleukin 8; hTNF-α: human tumor necrosis factor α; MAPKs: mitogen-activated protein kinases; NF-κB: nuclear factor “kappa-light-chain-enhancer” of activated B cells.
Figure 8: AF1 and AF2 initiate MyD88 signaling independently of TLR5. (Fig. 8a, b, c, d) THP-1 cells (10 ⁵ /well) (a, b) and HDF (10 ⁴ /well) (c, d) were treated with various concentrations (1.5, 3 or 6 μM) of TH1020. was pretreated for 1 hour and post-treated with the indicated concentrations of FLA-ST, AF1 or AF2. After 24 hours post-treatment, culture supernatants of treated THP-1 cells were analyzed to determine levels of hIL-8 (a) and hTNF-α (b), and culture supernatants of HDFs were analyzed by ELISA to determine hIL-8 (a) and hTNF-α (b) levels. 8(c) and hIL-6(d) levels were evaluated. (FIG. 8E) TH1020 (6 μM)-mediated reduction in NF-κB and MAPK phosphorylation during AF1 (5 μM) treatment was analyzed by Western blotting of THP-1 cell lysates at the indicated time points. (FIG. 8f, g) RAW 264.7 cells (10 ⁵ /well) were treated with the indicated concentrations of PAM3CSK4, LPS, FLA-ST, AF1 or AF2. After 24 hours of treatment, culture supernatants were analyzed by ELISA to quantify the secretion of murine TNF-a (mTNF-α) (f) and mIL-6 (g). (FIG. 8H) The level of hIL-8 secretion by HEK-hTLR5 cells (3×10 ⁴ /well) was determined by ELISA after 24 hours of treatment with the indicated concentrations of the peptide and FLA-ST. The experiment was independently performed 4 times, and the mean ± SEM difference between the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05). FLA-ST: flagellin from S. Typhimurium; hIL-6: human interleukin 6; hIL-8: human interleukin 8; hTNF-α: human tumor necrosis factor α; MAPKs: mitogen-activated protein kinases; NF-κB: nuclear factor “kappa-light-chain-enhancer” of activated B cells.
Figure 9: AF1 and AF2 activate signaling through TLR1/2 and TLR4. (FIG. 9a) RAW 264.7 cells (10 ⁵ /well) were pretreated for 1 hour with the indicated concentrations of Cu-CPT22, TAK-242 or both. Post-treatment for 24 hours with the indicated concentrations of AF1 and AF2. Culture supernatants were collected to quantify the secretion of mTNF-α by ELISA. (FIG. 9B) The level of hIL-8 secretion by HEK-hTLR2 cells (3×10 ⁴ /well) was measured by ELISA 24 hours after treatment with the indicated concentrations of the peptides, PAM3CSK4 and FSL-1. (FIG. 9c) SEAP activity of HEK-Blue™ hTLR4 cells (3×10 ⁴ /well) was measured using the SEAP Reporter Assay Kit 24 hours after treatment with the indicated concentrations of peptides, PAM3CSK4, FSL-1, FLA-ST or LPS. decided. (FIG. 9D) IL-8 secretion levels were determined after 24 hours of treatment of 293/hTLR2-TLR6 cells with the aforementioned reagents. (FIG. 9e) RAW 264.7 cells (10 ⁵ /well) were pre-treated with L48H37 for 1 hour and post-treated with the indicated concentrations of the compound for 24 hours. Then, the secretion level of TNF-α was determined by ELISA. The experiment was independently performed 4 times, and the mean ± SEM difference between the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05). FLA-ST: flagellin from S. Typhimurium; mTNF-α: mouse tumor necrosis factor α; mIL-6: mouse interleukin 6; SEAP: secreted embryonic alkaline phosphatase.
Figure 10: Immunofluorescence analysis shows the specificity of AF1 for TLR2 and TLR4. (FIG. 10a, b) RAW 264.7 cells (a) and HDFs (b) were grown overnight in 24-well plates. Next, cells were pretreated with the indicated concentrations of Cu-CPT22, TAK-242, both or DMSO. Post-treatment was based on AF1 (5 μM), and immunofluorescence analysis was performed using specific anti-p-p65 and anti-β-actin primary antibodies and Alexa Fluor-conjugated secondary antibodies. Red color represents phosphorylated NF-κB (p-p65), green color represents β-actin, and blue staining by Hoechst 33258 dye represents the nucleus. Images were captured at ×40 magnification using an inverted microscope (Olympus IX53; Olympus Corporation, Tokyo, Japan).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

Facultative or opportunistic pathogenic bacteria cause disease after entering the host when the host is in a severely immunocompromised and debilitated state (Parke, JL et al., Annual review of phytopathology ). 2001, 39 , 225-258). Among them, a special group of bacteria causes nosocomial infections known as nosocomial infections. For example, 45% of patients in intensive care units in Europe have been reported to be infected with opportunistic pathogens (Vincent, J.-L. et al., Jama 1995, 274 , 639-644). A. avenae is a phytopathogenic bacterium that infects various crops such as rice, maize, sugar cane, and oats (Lipuma, JJ et al., In Manual of Clinical Microbiology, Eleventh Edition , American Society of Microbiology: 2015; pp. 791 -812). In addition to plants, fever, blood cancer, sepsis and other bloodstream infections have been found in human patients (Malkan, AD et al., Journal of clinical microbiology 2009, 47 , 3358-3361). However, the immunomodulatory action at the molecular level in humans has not been investigated so far.

In plants, induction of PAMP-induced immunity by flagellin (named 'FLA-AA') isolated from rice-toxic strain A. avenae N1141 has been reported. Based on sequence similarity to human pathogenic bacteria, it was hypothesized that purified flagellin could induce immune signaling at the cellular and molecular level in humans during opportunistic infections. PAMPs are initially recognized by PRRs of the innate immune system, and TLRs are some of the PRRs that can recognize their cognate ligands and initiate TLR signaling. Among extracellular TLRs, TLR5 is a homodimer that recognizes the flagellin protein of pathogenic bacteria. Ligand recognition allows TLRs to interact with cytoplasmic adapter molecules and downstream signaling molecules for activation of transcription factors such as NF-κB, AP-1 and IRF (Ve, T. et al., Current drug targets 2012, 13 , 1360-1374). The present inventors confirmed that FLA-AA has antigenic properties for human macrophages and fibroblasts at the molecular level in terms of secretion of pro-inflammatory cytokines (FIG. 2). The specificity of FLA-AA was confirmed using a TLR5 inhibitor (TH1020) and a TLR5 overexpressing cell line (293/hTLR5), and it was confirmed that FLA-AA-induced signaling was mediated by TLR5 (FIG. 3). In addition, the TLR5-mediated activation of the signaling pathway by FLA-AA was supported by the results of immunofluorescence analysis (FIG. 4).

The inflammasome is a multiprotein cytoplasmic complex found in macrophages and dendritic cells that activates caspase 1 to induce inflammatory cell death (pyroptosis) and release of mature IL-1β and IL-18. (Martinon, F. et al., Molecular cell 2002, 10 , 417-426). Various cytoplasmic PRRs mediate inflammasome assembly and subsequent inflammasome-based innate immunity (Miao, EA et al., Nature immunology 2010, 11 , 1136). Among them, NLRC4 is a member of the NLRC subfamily of NLRs reported to recognize bacterial flagellin. The bacterial flagellin protein contains two evolutionarily conserved leucine residues at the C-terminus, which are common in many substrates of the type IV secretion system (Nagai, H. et al., Proceedings of the National Academy of Sciences). Sciences 2005, 102 , 826-831). They collectively contribute to the cytoplasmic delivery of flagellin for caspase 1 activation. by infection or by S. Typhimurium, Legionella pneumophila , Pseudomonas aeruginosa It has been reported that NLRC4 inflammasome can be triggered by purified flagellin of mammalian pathogenic bacteria such as Listeria monocytogenes and Listeria monocytogenes (Warren, SE et al., The Journal of Immunology 2008, 180 , 7558-7564, etc.). In the present invention, it was confirmed that flagellin purified from the plant pathogenic bacterium A. avenae activates caspase 1 to release mature IL-1β (FIG. 2).

The N and C termini of flagellin constitute the D0/D1 domain, which has been reported to play an important role in the binding and activation of TLR5 to downstream signaling pathways (Murthy, KG et al., Journal of Biological Chemistry 2004 , 279 , 5667-5675). Therefore, the present inventors selected one section (AF1) of amino acid residues in the C-terminal D0/D1 domain and three sections (AF2, AF3, and AF4) in the N-terminal D0/D1 domain (FIG. 5). Due to the length of the effective region, none of the peptides showed agonistic activity on TLR5. A TLR5-specific recombinant agonist, entolimod (CBLB502), was developed by fully connecting the N and C termini of flagellin via a flexible linker, pharmacologically optimized for reduced immunogenicity with high specificity (Burdelya, LG et al. ., Science 2008, 320 , 226-230). The N terminus of flagellin forms two α-helices. The first α-helix consists of amino acid residues 57-99 and the second consists of residues 104-129. The C terminus, on the other hand, forms a single α-helix (residues 406-447) equal to the length of the first α-helix of the N terminus (Samatey, FA et al., Nature 2001, 410 , 331-337). These helices are the main structures responsible for the function of flagellin. The 14 amino acid "motif N" (residues 95-108) in the N-terminus is very important as its deletion abolishes flagellin's pro-inflammatory activity. Peptides AF1 (residues 408-443) and AF2 (residues 85-120), despite containing most of the residues of the C- and N-terminal α-helices, respectively, initiate pro-inflammatory signaling independent of TLR5. This may be because some residues before the 85th from the N-terminus or after residue 443 as the 9 amino acid “motif C” (residues 441-449) within the C-terminus are functionally essential for TLR5 activation (Murthy, KG et al. , Journal of Biological Chemistry 2004, 279 , 5667-5675). Peptides AF3 (residues 65-96) and AF4 (residues 31-48 + 90-101) do not activate pro-inflammatory signaling as they contain residues 88-97 known to bind to the TLR5 site composed of residues 552-561. (Jacchieri, SG et al., Journal of bacteriology 2003, 185 , 4243-4247). We hypothesized that they bind to TLR5 without triggering signaling and inhibit signaling by blocking the binding of full-length flagellin.

In the present invention, it was confirmed that flagellin-derived peptides can be recognized by TLRs other than TLR5. This result suggests that some TLRs can recognize uncommon ligands despite their high specificity for major PAMPs:

(1) TLR4 recognizes LPS in a sequential manner: LPS forms a complex with CD14 followed by an LPS-binding protein that is recognized by TLR4 itself. MD-2 enhances LPS responsiveness by binding to the extracellular domain of TLR4. TLR4 has been reported to recognize ligands other than LPS, such as taxol, heat shock protein (HSP) 60, fibronectin, hyaluronic acid, heparin sulfate, and fibrinogen ( Smiley, ST et al., The Journal of Immunology 2001, 167 , 2887-2894, etc.).

(2) Together with TLR1 and TLR6, TLR2 activates lipoproteins, peptidoglycan and lipoteichoic acid, lipoarabinomanan, glycoinositol phospholipids, and zymosan. zymosan, glycolipids and porins (Aliprantis, AO et al., Science 1999, 285 , 736-739, etc.).

In the present invention, it was confirmed that peptides recognized by TLR4 are also recognized by TLR1/2 and initiate downstream signaling pathways. This indicates that a ligand specific for one TLR can also be recognized by other extracellular or intracellular molecules.

Accordingly, in one aspect, the present invention relates to a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by a peptide bond. The peptide may be prepared according to a chemical synthesis method known in the art.

In the present invention, the amino acid sequences of flagellin (FLA-AA) isolated from the plant pathogenic bacterium A. avenae N1141 and peptide fragments AF1 to AF4 derived therefrom are shown in Table 1 below.

name amino acid sequence sequence number AF1 IDAALSAVNGQRASFGALQSRFETTVNNLQSTSEN One AF2 GDILQRVRELAVQSANATNSSGDRKAIQAEVGQLL 2 AF3 VRNANDGISLAQTAEGALKSTGDILQRVRELA 3 AF4 RLSSGLRINSAKDDAAGLQRVRELAVQSAN 4 FLA-AA MASTINTNVSSLTAQRNLSLSQSSLNTSIQRLSSGLRINSAKDDAAGLAISERFTSQIRGLNQAVRNANDGISLAQTAEGALKSTGDILQRVRELAVQSANATNSSGDRKAIQAEVGQLLSEMDRIAGNTEFNGQKLLDGSFGSATFQVGANANQTITATTGNFRTNNYGAQLTASATGAATTGATAGSAGAAAGTVVIAGLQTKTVNVAA AGTASDIASAVNAVADSTGVTASARNVSEMKFSGTGSFTLAVKGDNSTAANVTFNVSATSTAAGLAEAVKAFNDVSSQTGVTAKLNSDSSGLILTNESGNDINIANGSSSAAGITLASQDAVTTQSSGTLTFTSATAAGTGVTVASRGTVEYKSDKGYTVSGTGGTMTNATATSSTLTKVSDIDVSTVDGSTKALKIIDAALSAVNGQRASFGALQSR FETTVNNLQSTSENMSASRSRIQDADFAAETANLSRSQILQQAGTAMVAQANQLPQGVLSLLK 5

In the present invention, the peptide may be characterized by activating TLR (Toll-like receptor) 1/2 and/or TLR4 signaling pathways.

As used herein, the term “TLR1/2 signaling pathway” refers to a signaling pathway through TLR1 and/or TLR2, and may be a lipopeptide reaction dependent on the TLR1/TLR2 complex, a transmembrane complex formed by TLR1 and TLR2. , through which a signal is transmitted.

“TLR2” uniquely forms heterodimers with TLR1 to recognize a wide range of pathogenic components, such as Pam ₃ CSK ₄ , Pam ₂ CSK ₄ , lipotoichoic acids, zymosan, bacterial and fungal lipids, acylated sugars, and proteins. Activated TLR2 recruits MyD88 protein, and MyD88 recruits interleukin-receptor-associated kinase 4 (IRAK4), which activates IRAK1 through phosphorylation.

In the present invention, the term “TLR4” refers to a protein belonging to TLRs, a transmembrane protein family that functions as a monitor for pathogen infection, and refers to a protein encoded by the TLR4 gene and is designated as CD 284 (cluster of differentiation 284). are also named The TLR4 is very important for activation of the innate immune system because it recognizes various PAMPs including LPS of Gram-negative bacteria.

As used herein, the term “TLR4 signaling pathway” refers to a signaling pathway through TLR4, and may be an LPS response dependent on the TLR4/MD2 complex, a transmembrane complex formed by TLR4 and MD2, through which signals are transmitted. do. TLR4 signals by several adapter proteins, and the signaling pathways operate as Mal (also referred to as TIRAP) and MyD88, and TRAM and TRIF. The TLR4-mediated signaling pathway induces the activation of MyD88-dependent and MyD88-independent signaling through various downstream signaling molecules. Initiation of the MyD88-dependent pathway leads to early-stage activation of NF-κB and secretion of pro-inflammatory cytokines such as TNF-α and IL-6, and initiation of the MyD88-independent pathway leads to activation of IRF3 and 7, IFNs secretion and late phase activation of NF-κB. In addition, TLR4 induces activation of MAPKs including ERK, JNK and p38, and secretes inflammatory cytokines and IFNs. In macrophages, stimulation of TLR4 by related ligands induces COX2 and iNOS, induces NO production, and generates mitochondrial and intracellular ROS.

In the present invention, the peptide may be characterized by inducing activation of nuclear factor kappa B (NF-κB) or phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, the peptide may be characterized by inducing the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), or interleukin-8 (IL-8).

In one embodiment of the present invention, the pro-inflammatory cytokine secretion effect through activation of the TLR signaling pathway of AF1 and AF2 was confirmed.

Accordingly, from another aspect, the present invention relates to a Toll-like receptor (TLR) agonist comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the term “TLR agonist” activates TLRs and induces, in particular, biological responses.

Agonists or antagonists (antagonists) of TLRs have been shown to target the same site on the receptor (Manavalan B, Basith S, & Choi S (2011) Frontiers in physiology 2:41). Thus, activation or inhibition of a particular TLR may depend on how much steric strain a given substance imparts upon binding. For example, the bacterial immune evasion protein staphylococcus super antigen-like 3 (SSL3) has been shown to target the lipid peptide binding cavity of TLR2 and dominates immune signaling independent of the agonist bound (Koymans KJ , et al . al (2015) Proceedings of the National Academy of Sciences of the United States of America 112(35):11018-11023). It has been reported that small molecule antagonists such as CU-CPT22 occupy an extended hydrophobic cavity in TLR1 channels and inhibit TLR1/2 activation upon agonist stimulation.

In the present invention, the TLR may be TLR1/2 and/or TLR4, but is not limited thereto.

In the present invention, AF1 and AF2 may directly or indirectly, or substantially induce, increase or trigger the biological activity of TLR1/2 and/or TLR4. Thus, the TLR agonist may be characterized as a TLR1/2 and/or TLR4 agonist.

TLRs are attractive targets for vaccine adjuvants because of their ability to activate both the innate and adaptive immune systems. It has been reported that TLR2 agonists can be effective adjuvants for the treatment of viral infections, and lipopeptide mimetic agonists of TLR2 have been reported to show strong adjuvant effects with various antigens. In addition, the weakened TLR1/2 signal is related to infection-related disease morbidity due to aging.

Therefore, in another aspect, the present invention relates to an adjuvant comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Adjuvants strengthen the immune system by linking antigens to antigen-presenting cells or by inducing specific inflammatory responses to weak or non-immunogenic components (Coffman RL, et al. (2010) Immunity 33(4):492 -503). The immune response is initiated by TLRs through a signaling cascade, resulting in NF-κB-mediated transcription of pro-inflammatory cytokines and chemokines.

TLR agonists have shown promising results in reducing lung tumors, bladder cancer, breast cancer, and pancreatic ulcers, and TLR1/2 agonists, apart from anticancer drugs, are effective in treating various infectious diseases such as chronic and acute influenza, asthma, and age-related obesity. It has been reported that

Accordingly, in another aspect, the present invention relates to a composition for preventing or treating cancer comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In another aspect, the present invention relates to a method for preventing or treating cancer comprising administering the peptide.

In another aspect, the present invention relates to the use of the peptide for preventing or treating cancer.

In another aspect, the present invention relates to the use of the peptide for preparing a drug for preventing or treating cancer.

In the present invention, the cancer is used in the same sense as a tumor, and is selected from the group consisting of lung tumor, bladder cancer, cervical cancer, melanoma, prostate cancer, liver cancer, lymphoma, colon cancer, ovarian cancer, breast cancer and pancreatic cancer. It can be characterized, but is not limited thereto.

The term "prevention" used in the present invention refers to all activities that inhibit or delay cancer by administering the peptide. In addition, the term "treatment" used in the present invention refers to all activities in which cancer symptoms are improved or cured by administration of the peptide.

The composition for preventing or treating cancer according to the present invention may contain a pharmaceutically effective amount of the peptide alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmacologically effective amount in the above refers to an amount sufficient to prevent, improve, and treat symptoms of cancer.

The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives may be further included.

The term "carrier" refers to a substance that facilitates the addition of a compound into a cell or tissue. The term "diluent" is defined as a substance that is diluted in water that not only stabilizes the biologically active form of the subject compound, but also dissolves the compound.

In addition, the composition of the present invention may include one or more known active ingredients having a therapeutic effect on cancer together with the peptide.

The compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a non-human mammal. The dosage form may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.

The composition of the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on various factors such as the route of administration, age, sex, weight and severity of the patient. The composition according to the present invention may be appropriately selected depending on the condition, and the composition according to the present invention may be administered in combination with a known compound having an effect of preventing, improving, or treating symptoms of cancer.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating TLR1/2 and/or TLR4 related diseases comprising the peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In another aspect, the present invention relates to a method for preventing or treating a TLR1/2 and/or TLR4 related disease comprising administering the peptide.

In another aspect, the present invention relates to the use of the peptide for preventing or treating TLR1/2 and/or TLR4 related diseases.

In another aspect, the present invention relates to the use of the peptide for the preparation of a drug for preventing or treating TLR1/2 and/or TLR4 related diseases.

In the present invention, the TLR1/2 and/or TLR4-related disease may be selected from the group consisting of lime disease, chronic or acute influenza infection, and viral disease, but is not limited thereto.

In the present invention, the viral disease means a viral disease that includes all virus groups consisting of ssRNA, dsRNA, dsDNA or ssDNA. ssRNA viruses include astroviridae (positive-sense ssRNA; human astrovirus), caliciviridae (positive-sense ssRNA; norovirus), and picornaviridae (positive-sense ssRNA; coxsackievirus, hepatitis A, poliovirus, rhinovirus) , Coronavirus (positive-sense ssRNA; severe acute respiratory syndrome virus, SARS-CoV-2), Flaviviridae (positive-sense ssRNA; Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus TBE virus), To Positive-sense ssRNA (rubella virus), positive-sense ssRNA (Hepatitis E virus), retroviridae (ssRNA-RT; human immunodeficiency virus HIV), orthomyxoviridae (negative-sense ssRNA; orthomyxo) Virus), arenaviridae (negative-sense ssRNA; Lassa virus), buniaviridae (negative-sense ssRNA; Crimean-Congo hemorrhagic fever, Hantaan virus), filoviridae (negative-sense ssRNA; Ebola virus, Marburg virus), paramyxo Viridae (negative-sense ssRNA; Measles virus, Mumps virus, Parainfluenza virus, respiratory syncytial virus), Rabdoviridae (negative-sense ssRNA; Rabies virus), Hepatitis D (negative-sense ssRNA) are included, and dsRNA viruses There are reoviridae (dsRNA; rotavirus, Orbivirus, Coltivirus, Banna virus), and dsDNA viruses include adenoviridae (dsDNA; adenovirus), herpes simplex virus (dsDNA; herpes simplex type 1, herpes simplex type 2, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, KSHV), Papillomaviridae (dsDNA; human papillomavirus), polyomaviridae (dsDNA; BK virus, JC virus), poxviridae (dsDNA; smallpox), and hepadnaviridae (dsDNA-RT; hepatitis B virus), and ssDNA viruses include Parvoviridae (ssDNA; parvovirus B19), and may be characterized in that it is a disease selected from viral diseases caused by this group of viruses, but is not limited thereto.

Examples of viral diseases include, but are not limited to, cold, flu (influenza), chickenpox, herpes zoster, herpes simplex, infectious mononucleosis, cytomegalovirus infection, measles, mumps, rubella, parvovirus infection, polio (polio), virus Includes hemorrhagic fever, yellow fever, dengue fever, rabies, AIDS and Covid-19.

Since the pharmaceutical composition for preventing or treating TLR1/2 and/or TLR4-related diseases includes a pharmaceutical formulation containing the above-described peptide, descriptions overlapping with the above-described composition of the present invention will be omitted.

Other terms and abbreviations used in this specification may be interpreted as meanings commonly understood by those skilled in the art to which the present invention belongs unless otherwise defined.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Materials and Methods

Example 1-1: cell lines and reagents

THP-1 cells (obtained from Prof. Changhee Seo, Ajou University Medical Center (Suwon, Korea)), A549 cells (ATCC, Manassas, VA, USA) and MDA-MB-231 cells (obtained from Prof. Kyungsook Choi, Ajou University Medical Center (Suwon, Korea)) ) were cultured in RPMI1640 supplemented with 1% penicillin/streptomycin solution and 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA). THP-1 cells were differentiated into macrophages using 80 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich Co., St. Louis, MO, USA) for 24 hours. human dermal fibroblasts (HDF; ATCC); 293/hTLR5, 293/hTLR2 and HEK-Blue™ hTLR4 cells (InvivoGen, San Diego, Calif., USA); and MCF-7 231 cells (obtained from Professor Kyungsook Choi) in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin/streptomycin solution, 10% FBS and 0.2% normocin. ) was cultured in RAW 264.7 cells (macrophages; Korea Cell Line Bank, Seoul, Korea) were cultured in high-glucose DMEM containing 1% penicillin/streptomycin solution and 10% FBS (Thermo Fisher Scientific, Inc.). All cells were cultured under 5% CO ₂ , 37° C. humid conditions (Thermo Fisher Scientific, Inc.). Lipofectamine 2000 and PAM ₃ CSK ₄ were purchased from Thermo Fisher Scientific, Inc., FSL-1 and FLA-ST were purchased from InvivoGen, TH1020 was purchased from Tocris (Cookson, Bristol, UK), LPS ( Escherichia coli 0111:B4), Cu-CPT22, TAK-242 and PMA were purchased from Sigma-Aldrich Co. Flagellin from A. avenae (FLA-AA) was purified according to a previously described protocol (Hirai, H. et al., Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin. 2016). All peptides used in the experiment were synthesized by BioStem (Ansan, Korea).

Example 1-2: cell viability assay

Cell viability was calculated by performing colorimetric 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay (Sigma-Aldrich Co.). THP-1 cells (10 ⁵ /well), HDFs (10 ⁴ /well), A549 cells (10 ⁴ /well), MCF-7 cells (1.5×10 ⁴ /well) and MDA-MB-231 cells (1.5×10 4 /well). 10 ⁴ /well) was dispensed into a 96-well plate (BD Biosciences, San Jose, CA, USA) and grown overnight. Cells were treated with various concentrations of FLA-ST, FLA-AA or AF1-4 for 24 hours. The next day, the medium was replaced with a medium containing 10% MTT solution (100 μl/well) and incubated at 37° C. for 3 hours. Replaced with DMSO (100 μl/well) and incubated the plate at 37° C. for 30 minutes. Plates were read at 540 nm wavelength using a microplate spectrophotometer system (Molecular Devices, Silicon Valley, California).

Example 1-3: SEAP (secreted embryonic alkaline phosphatase ) activity assay

HEK-Blue™ hTLR4 cells (3×10 ⁴ /well) were grown overnight in 96-well plates (BD Biosciences). Cells were treated with various concentrations of AF1, AF2, PAM3CSK4, FSL-1, LPS or FLA-ST for 24 hours. The collected culture supernatant (200 μl) was heated on a heating block (FINEPCR Co., Seoul, Korea) at 65°C for 10 minutes, and then transferred to a new 96-well plate (BD Biosciences) using the SEAP Reporter Assay Kit (InvivoGen). SEAP production was detected. Absorbance was measured at 620 nm with a microplate reader spectrophotometer system (Molecular Devices Inc., Silicon Valley, CA, USA).

Example 1-4: western blot (western blot) analysis

Total cell protein was extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc.), and protein concentration was measured with a Bicinchoninic Acid (BCA) Assay Kit (Sigma-Aldrich). Protein samples were electrophoresed and transferred to the membrane of a Mini-PROTEAN Tetra Cell and Mini Trans-Blot Electrophoretic Transfer Cell System (Bio-Rad Laboratories, Hercules, CA, USA). phospho-(p-)p65, p-ERK, ERK, JNK, p-JNK, p38, p-p38, Iκ-Bα (Cell Signaling Technology Inc., Danvers, MA; USA) and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) were immunoblotted with specific primary antibodies (1:500-1:1000 dilution). After thorough washing in PBS supplemented with 0.1% Tween 20, the membrane was incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (1:1,000) for 2 hours at room temperature. Protein bands were detected using SuperSignal West Pico ECL Solution (Thermo Fisher Scientific, Inc.) and visualized using the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories).

Example 1-5: immunofluorescence ( Immunofluorescence )

THP-1 cells, RAW 264.7 cells or HDFs were grown on cover slips in 24-well plates (BD Biosciences) at a density of 10 ⁴ /well. THP-1 cells and HDFs were either pretreated with TH1020 (3 μM) for 1 h before treatment with FLA-ST or FLA-AA (200 ng/ml), or not. Similarly, RAW 264.7 cells and HDFs were either pretreated with Cu-CPT22 (20 μM), TAK-242 (20 μM), or both, or DMSO for 1 h before stimulation with AF1 (5 μM), or not. Then, the cells were fixed, permeabilized with cold methanol (Samchun Chemicals, Korea) for 10 minutes, washed with PBS, and blocked with PBS (Thermo Fisher Scientific, Inc.) containing 3% BSA solution for 30 minutes. Cells were incubated overnight at 4° C. with anti-p-p65 antibody (1:1000; Cell Signaling Technology Inc.) and anti-β-actin antibody (1:1000; Santa Cruz Biotechnology Inc.). After thorough washing, cells were incubated with Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. After washing with PBS, nuclei were stained with Hoechst 33258 solution (5 μM; Sigma-Aldrich) for 10 minutes. Images were captured using a fluorescence microscope (Olympus IX53; Olympus Corporation, Tokyo, Japan).

Example 1-6: cytokines ( cytokines ) detection assay

THP-1 derived macrophages (10 ⁵ /well), HDFs (10 ⁴ /well), 293/hTLR5 cells (3×10 ⁴ /well), A549 cells (10 ⁴ /well), MCF-7 cells (1.5× 10 ⁴ /well), MDA-MB-231 cells (1.5×10 ⁴ /well), 293-hTLR2 cells (3×10 ⁴ /well) and RAW 264.7 cells (10 ⁵ /well) were plated in a 96-well plate (BD Biosciences) and grown overnight. After 24 hours of treatment, TNF-α production was measured by Human and Mouse TNF Alpha Uncoated ELISA Kit (eBioscience, San Diego, CA, USA), and IL-6 was measured by Human IL-6 ELISA MAX Deluxe Kit (BioLegend, San Diego, CA, USA). USA), IL-8 was evaluated with Human IL-8 Uncoated ELISA Kit (eBioscience). For NLRC4 activation, THP-1 macrophages were primed with LPS (1 μg/ml) for 4 hours and then transfected with FLA-AA and FLA-ST for 2 hours using Lipofectamine 2000. To evaluate the amount of secretion of IL-1β, the culture supernatant was collected using the IL-1 beta Human Uncoated ELISA Kit (Thermo Fisher Scientific, Inc.). Plates were then analyzed with a microplate spectrophotometer system (Molecular Devices) at each wavelength.

Example 1-7: Statistical analysis

The data presented are the average of 4 independent experiments, and the difference between the mean ± SEM of the experimental groups was evaluated by two-tailed paired Student's t test (*P < 0.05).

Example 2: Immune signaling in human macrophages and fibroblasts activating FLA-AA

Prior to functional analysis of FLA-AA, through TLR5 (Hayashi, F. et al., Nature 2001, 410 , 1099-1103) and NLRC4 (Zhao, Y. et al., Nature 2011, 477 , 596-600) Sequence similarity with flagellin protein extracted from Salmonella typhimurium , which has been reported to activate the human immune system, was confirmed. Using "EMBOSS water", a pairwise local sequence alignment tool of EMBL-EBI, 40.1% identity between sequences was confirmed (Fig. 1).

PAMPs are recognized by various host PRRs, such as TLRs, and can dimerize for subsequent activation of downstream signaling pathways. The initiated signaling pathway induces the secretion of various cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 8 (IL-8) (Akira, S. et al., Nature reviews immunology 2004, 4 , 499). Evaluation of toxicity and immunogenicity of FLA-AA and FLA-ST (positive control) with respect to activation of immune signaling pathways in PMA-differentiated human macrophages (THP-1 cells) and fibroblasts (HDF) did

The toxicity profile of FLA-AA was consistent with that of FLA-ST on THP-1 cells (toxic at high concentrations; Fig. 2a) and HDFs (no toxicity; Fig. 2d). In addition, dose-dependent secretion of IL-8 (FIG. 2B) and TNF-α (FIG. 2C) by THP-1 cells was observed after 24 hours of treatment. Similarly, dose-dependent secretion of IL-8 (FIG. 2E) and IL-6 (FIG. 2F) by HDFs was detected after treatment with either FLA-AA or FLA-ST for 24 hours. Ligand recognition by TLRs activates downstream NF-κB and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. Therefore, after treating human macrophages (THP-1 cells) with FLA-AA, activation of NF-κB (phosphorylation of p65) and MAPKs (phosphorylation of ERK, JNK, and p38) was observed in a time-dependent manner (Fig. 2g).

When cells primed with PAMP recognize intracellular flagellin protein, they activate NLRC4, which helps caspase 1 to release inflammatory cytokines such as IL-1β and IL-18 (Miao, EA et al., Nature immunology 2006, 7 , 569). Considering the ability of flagellin proteins to activate NLRC4, it is possible that flagellins derived from plant pathogens (FLA-AA) can stimulate NLRC4 in human cells as well as those derived from human pathogens (e.g. FLA-ST). It was tested whether there is Lipofectamine 2000 was used for intracellular delivery of FLA-AA or FLA-ST, and significant secretion of IL-1β was confirmed in a dose-dependent manner using a mixture of FLA-AA and Lipofectamine 2000. However, IL-1β was not detected when treated with FLA-AA alone (Fig. 2h).

Taken together, it suggests that FLA-AA is immunogenic in terms of initiating the TLR signaling pathway and triggering NLRC4 upon target cell processing.

Example 3: to TLR5 Specific FLA-AA-mediated signaling

Flagellin, a major component of bacterial flagella, has an evolutionarily conserved region recognized by TLR5 expressed in a variety of cells (Gewirtz, AT et al., The Journal of Immunology 2001, 167 , 1882-1885). . The small molecule TH1020 competes with flagellin for binding to TLR5, acting as a TLR5 inhibitor (Yan, L. et al., ChemMedChem 2016, 11 , 822-826).

To evaluate the specificity of FLA-AA for TLR5 in THP-1 cells (Figures 3a and 3b) and HDFs (Figures 3c and 3d), FLA-AA (200ng/ml) or FLA-ST (200ng/ml) and Together, cells were treated with various concentrations of TH1020 (1.5, 3 or 6 μM), and the concentration of secreted cytokines was examined. In THP-1 cells, a TH1020-dependent decrease in IL-8 (FIG. 3A) and TNF-α (FIG. 3B) secretion was observed, whereas in HDFs, IL-8 (FIG. 3C) and IL-6 (FIG. 3D) were decreased. A decrease in secretion was observed.

Next, FLA-AA toxicity and signaling activation were evaluated in a TLR5 overexpressing cell line (293/hTLR5). There was no significant toxicity during FLA-AA treatment up to 500 ng/ml (Fig. 3e), and significant secretion of IL-8 was observed 24 h after treatment (Fig. 3f). Activation of TLR5 signaling leads to phosphorylation of the p65 subunit of NF-κB, which can serve as an intracellular marker. After culturing FLA-AA (200 ng/ml) with THP-1 cells (FIG. 4a) and HDFs (FIG. 4b), phosphorylation of p65 (p-p65) was visualized using immunofluorescence. Consistent with previous results, the fluorescence intensity of p-p65 decreased in THP-1 cells (Fig. 4a) and HDFs (Fig. 4b) after co-treatment of cells with FLA-AA and the TLR5 inhibitor TH1020. Through these results, it can be confirmed that FLA-AA is specifically recognized by TLR5 to initiate a downstream signaling pathway.

Example 4: Derived from FLA-AA that activates immune signaling in human macrophages and fibroblasts peptide

Flagellin protein contains several domains, among which D0 and D1 have been reported to play important roles in binding and triggering TLR5 to downstream signaling pathways (Yoon, S.-i. et al., Science 2012 , 335 , 859-864). Therefore, one section (AF1) of amino acid residues was selected in the C-terminal D0/D1 domain, and three sections (AF2, AF3, and AF4) were selected in the N-terminal D0/D1 domain (FIG. 5).

First, along with FLA-ST (positive control), the toxicity and immunogenicity of the peptides AF1-AF4 on PMA-differentiated human macrophages (THP-1) and HDFs were evaluated. To THP-1 cells, peptides AF1 and AF2 were slightly toxic at high concentrations, while AF3 and AF4 were non-toxic (Fig. 6a), while all peptides (AF1-AF4) were toxic to HDFs at high concentrations (Fig. 6b). ). After treatment of THP-1 cells (Figs. 7a and 7b) and HDFs (Figs. 7c and 7d) with each peptide for 24 hours, dose-dependent secretion of IL-8 was observed only from peptides AF1 and AF2 (Figs. 7a and 7c). . Also, AF1 and AF2 induced dose-dependent secretion of TNF-α (FIG. 7B) and IL-6 (FIG. 7D). Recognition of ligands by TLRs activates downstream NF-κB and MAPKs, including JNK and p38. Since AF1 was more immunogenic than AF2, it was selected for analysis of downstream signaling pathways. As expected, time-dependent activation of NF-κB (phosphorylation of p65 and degradation of IKBα) and MAPKs (phosphorylation of p38 and JNK) was observed after treatment with AF1 (Fig. 7e).

Taken together, these results suggest that AF1 and AF2 are immunogenic in terms of initiating the TLR signaling pathway upon culture with target cells.

Example 5: with TLR5 independently TLR AF1 and AF2 stimulate signaling pathways

Bacterial flagella are composed primarily of flagellin proteins recognized by TLR5 due to evolutionarily conserved regions. Since the peptides AF1 and AF2 were derived from flagellin of the A. avenae bacterium, we tested whether they activate TLR signaling through TLR5. TH1020 inhibits TLR5 signaling by competing with flagellin for binding to TLR5.

To test TLR5 specificity in THP-1 cells (Figures 8A and 8B) and HDFs (Figures 8C and 8D), various concentrations of TH1020 (1.5, 3 or 6 μM) and AF1 or AF2 (5 μM) were incubated with the cells. Thus, inhibition of cytokine secretion was investigated. A TH1020 dependent decrease in IL-8 (FIG. 8A) and TNF-α (FIG. 8B) secretion was observed in THP-1 cells. Considering the low potency of AF2, we performed the same experiment in HDFs with AF1 alone and observed a decrease in IL-8 (Fig. 8c) and IL-6 (Fig. 8d) secretion. We also observed TH1020 (6 μM)-dependent inhibition of the downstream TLR signaling pathway activated by AF1 (750 nM) ( FIG. 8e ). Since mouse macrophages (RAW 264.7 cells) do not express functional TLR5 (Means, TK et al., The Journal of Immunology 2003, 170 , 5165-5175), it was used as a negative control cell line for further validation. For reference, both peptides greatly enhanced the secretion of TNF-α (Fig. 8f) and IL-6 (Fig. 8g). Consistent with this result, none of the peptides induced cytokine secretion after treatment with the TLR5 overexpressing cell line (293/hTLR5) (Fig. 8h). The results suggest that AF1 and AF2 activate TLR signaling independently of TLR5.

Example 6: In an MD-2 dependent manner TLR1 /2 and TLR4 AF1 and AF2 activating signaling through

After detecting the initiation of TLR5-independent signaling, we hypothesized that AF1 and AF2 interact with other extracellular TLRs, namely TLR1/2, TLR2/6 or TLR4. To test this hypothesis, the peptides AF1 and AF2 were incubated with TLR2 overexpressing cells (293/hTLR2; FIG. 9A) or TLR4 overexpressing cells (HEK-Blue™ hTLR4; FIG. 9B). In both cell lines, both peptides significantly stimulated signaling, that is, signaling through TLR2 and TLR4, but not through TLR5. TLR2 activates signal transduction by forming a heterodimer with its core receptors TLR1 and/or TLR6 (Triantafilou, M. et al., Journal of Biological Chemistry 2006, 281 , 31002-31011) . Therefore, it is important to determine which TLR2 heterodimer(s) are activated by the peptide.

To test TLR1/2, mouse macrophages (RAW 264.7 cells) were separately pretreated with a TLR1/2 inhibitor (Cu-CPT 22), a TLR4 inhibitor (TAK-242) or both. Post-treatment included AF1 (5 μM) or AF2 (10 μM) for 24 h. These inhibitors significantly reduced the secretion of the inflammatory cytokine TNF-α (FIG. 9c). These results were confirmed by immunofluorescence analysis of RAW 264.7 cells (FIG. 10A) and HDFs (FIG. 10B). These results provide clues to the involvement of TLR1/2 heterodimers with TLR4 in signaling.

To test TLR2/6, a TLR2/6 overexpressing cell line (293/hTLR2-TLR6) was treated with both peptides and an appropriate control ligand. Unlike the TLR2/6 ligand, FSL-1, none of the peptides induced cytokine secretion (FIG. 9d). Moreover, AF1 and AF2 activated signaling in an MD-2 dependent manner, as confirmed by the MD-2 inhibitor (L48H37) (FIG. 9e).

As a result, it can be confirmed that AF1 and AF2 activate TLR signaling through TLR1/2 and TLR4.

As above, specific parts of the present invention have been described in detail, and it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Toll-like receptor 1/2 and/or 4 Activating Peptides and Uses Thereof <130> P20-B218 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 35 <212> PRT <213> artificial sequence <220> <223> AF1 <400> 1 Ile Asp Ala Ala Leu Ser Ala Val Asn Gly Gln Arg Ala Ser Phe Gly 1 5 10 15 Ala Leu Gln Ser Arg Phe Glu Thr Thr Val Asn Asn Leu Gln Ser Thr 20 25 30 Ser Glu Asn 35 <210> 2 <211> 35 <212> PRT <213> artificial sequence <220> <223> AF2 <400> 2 Gly Asp Ile Leu Gln Arg Val Arg Glu Leu Ala Val Gln Ser Ala Asn 1 5 10 15 Ala Thr Asn Ser Ser Gly Asp Arg Lys Ala Ile Gln Ala Glu Val Gly 20 25 30 Gln Leu Leu 35 <210> 3 <211> 32 <212> PRT <213> artificial sequence <220> <223> AF3 <400> 3 Val Arg Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln Thr Ala Glu Gly 1 5 10 15 Ala Leu Lys Ser Thr Gly Asp Ile Leu Gln Arg Val Arg Glu Leu Ala 20 25 30 <210> 4 <211> 30 <212> PRT <213> artificial sequence <220> <223> AF4 <400> 4 Arg Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala 1 5 10 15 Gly Leu Gln Arg Val Arg Glu Leu Ala Val Gln Ser Ala Asn 20 25 30 <210> 5 <211> 492 <212> PRT <213> artificial sequence <220> <223> FLA-AA <400> 5 Met Ala Ser Thr Ile Asn Thr Asn Val Ser Ser Leu Thr Ala Gln Arg 1 5 10 15 Asn Leu Ser Leu Ser Gln Ser Ser Leu Asn Thr Ser Ile Gln Arg Leu 20 25 30 Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Ala Ile Ser Glu Arg Phe Thr Ser Gln Ile Arg Gly Leu Asn Gln Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Leu Lys Ser Thr Gly Asp Ile Leu Gln Arg Val Arg Glu Leu Ala 85 90 95 Val Gln Ser Ala Asn Ala Thr Asn Ser Ser Gly Asp Arg Lys Ala Ile 100 105 110 Gln Ala Glu Val Gly Gln Leu Leu Ser Glu Met Asp Arg Ile Ala Gly 115 120 125 Asn Thr Glu Phe Asn Gly Gln Lys Leu Leu Asp Gly Ser Phe Gly Ser 130 135 140 Ala Thr Phe Gln Val Gly Ala Asn Ala Asn Gln Thr Ile Thr Ala Thr 145 150 155 160 Thr Gly Asn Phe Arg Thr Asn Asn Tyr Gly Ala Gln Leu Thr Ala Ser 165 170 175 Ala Thr Gly Ala Ala Thr Thr Gly Ala Thr Ala Gly Ser Ala Gly Ala 180 185 190 Ala Ala Gly Thr Val Val Ile Ala Gly Leu Gln Thr Lys Thr Val Asn 195 200 205 Val Ala Ala Ala Gly Thr Ala Ser Asp Ile Ala Ser Ala Val Asn Ala 210 215 220 Val Ala Asp Ser Thr Gly Val Thr Ala Ser Ala Arg Asn Val Ser Glu 225 230 235 240 Met Lys Phe Ser Gly Thr Gly Ser Phe Thr Leu Ala Val Lys Gly Asp 245 250 255 Asn Ser Thr Ala Ala Asn Val Thr Phe Asn Val Ser Ala Thr Ser Thr 260 265 270 Ala Ala Gly Leu Ala Glu Ala Val Lys Ala Phe Asn Asp Val Ser Ser 275 280 285 Gln Thr Gly Val Thr Ala Lys Leu Asn Ser Asp Ser Ser Gly Leu Ile 290 295 300 Leu Thr Asn Glu Ser Gly Asn Asp Ile Asn Ile Ala Asn Gly Ser Ser 305 310 315 320 Ser Ala Ala Gly Ile Thr Leu Ala Ser Gln Asp Ala Val Thr Thr Gln 325 330 335 Ser Ser Gly Thr Leu Thr Phe Thr Ser Ala Thr Ala Ala Gly Thr Gly 340 345 350 Val Thr Val Ala Ser Arg Gly Thr Val Glu Tyr Lys Ser Asp Lys Gly 355 360 365 Tyr Thr Val Ser Gly Thr Gly Gly Thr Met Thr Asn Ala Thr Ala Thr 370 375 380 Ser Ser Thr Leu Thr Lys Val Ser Asp Ile Asp Val Ser Thr Val Asp 385 390 395 400 Gly Ser Thr Lys Ala Leu Lys Ile Ile Asp Ala Ala Leu Ser Ala Val 405 410 415 Asn Gly Gln Arg Ala Ser Phe Gly Ala Leu Gln Ser Arg Phe Glu Thr 420 425 430 Thr Val Asn Asn Leu Gln Ser Thr Ser Glu Asn Met Ser Ala Ser Arg 435 440 445 Ser Arg Ile Gln Asp Ala Asp Phe Ala Ala Glu Thr Ala Asn Leu Ser 450 455 460 Arg Ser Gln Ile Leu Gln Gln Ala Gly Thr Ala Met Val Ala Gln Ala 465 470 475 480 Asn Gln Leu Pro Gln Gly Val Leu Ser Leu Leu Lys 485 490

Claims (11)

delete delete delete delete A Toll-like receptor (TLR) agonist comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
6. The agent according to claim 5, wherein the TLR is TLR1/2 or TLR4.
An adjuvant comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
A pharmaceutical composition for preventing or treating cancer containing a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
The method of claim 8, wherein the cancer is selected from the group consisting of lung tumor, bladder cancer, cervical cancer, melanoma, prostate cancer, liver cancer, lymphoma, colon cancer, ovarian cancer, breast cancer, and pancreatic cancer. Therapeutic pharmaceutical composition.
A pharmaceutical composition for preventing or treating TLR1/2 or TLR4 related diseases containing a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2,
The pharmaceutical composition, characterized in that the TLR1/2 or TLR4 related disease is selected from the group consisting of lime disease, chronic or acute influenza infection and viral disease. delete

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