Composition and peptide having immune function
By designing truncated interferon peptides and combining them with buffers and excipients, the problems of short half-life and difficult purification of cytokines were solved, achieving stable immunomodulatory effects and reducing the side effects of traditional interferons.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AINOS INC
- Filing Date
- 2024-09-04
- Publication Date
- 2026-04-17
AI Technical Summary
In existing technologies, cytokines have short serum half-lives, limited efficacy, and are difficult to purify and store, making it difficult to effectively regulate immune responses.
Design truncated type I interferon (IFN) peptides containing specific domains such as helix A, helix B, helix C, helix D, helix E, and AB loop, and combine them with buffers, carriers, and excipients. Modify them by means of palmitoylation to improve their properties to enhance their binding to and penetration of cell membranes.
It achieves effective regulation of immune responses in vitro, reduces the side effects of cytokine therapy, provides stable immunomodulatory effects, and has interferon-like functions without cytotoxicity.
Smart Images
Figure CN121889418A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to compositions and peptides with immune function, and more particularly to compositions and peptides capable of regulating immune responses. Background Technology
[0002] Cytokines are small proteins (approximately 5 to 25 kDa) that are crucial for cell signaling. Due to their size, cytokines cannot cross the lipid bilayer of the cell to enter the cytoplasm; therefore, they typically exert their function by interacting with specific cytokine receptors on the surface of target cells. Cytokines have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators.
[0003] Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines can be produced by a wide variety of cell types, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Cytokines can be produced by more than one cell type. They act through cell surface receptors and are particularly important to the immune system; cytokines regulate the balance between humoral and cellular immune responses and can modulate the responses of maturation, growth, and specific cell populations. In complex pathways, some cytokines can enhance or inhibit the effects of others. Cytokines are important for health and disease, especially in host immune responses to infection, inflammation, trauma, sepsis, cancer, and reproduction.
[0004] Interferon (IFN) is a cytokine produced by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. IFN allows for communication between cells to initiate the immune system's protective defenses to eliminate pathogens or tumors.
[0005] However, due to the short serum half-life, limited efficacy, and difficulties in purification and storage of cytokines, there is still a demand in this field for proteins and / or peptides with cytokine-like functions, as well as for protein property improvements that can address the aforementioned deficiencies. Summary of the Invention
[0006] The present invention provides a composition for regulating an immune response, comprising: a truncated type I IFN, mainly composed of at least one domain selected from helix A, helix B, helix C, helix D, helix E and AB loop; and a buffer, a carrier, and / or an excipient.
[0007] The present invention also provides a composition for treating or preventing viral infections, comprising: a truncated type I IFN, mainly composed of at least one domain selected from helical A, helical B, helical C, helical D, helical E and AB loop; and a buffer, a carrier, and / or an excipient.
[0008] This invention provides a peptide for regulating immune responses, primarily composed of at least one amino acid sequence selected from the following: SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK; SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN; SEQ ID NO. 3: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*; C* indicates that the amino acid is modified by adding a disulfide bond; SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; and An amino acid sequence having at least 80% identity with any of SEQ ID NO. 1 to 4.
[0009] The present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the aforementioned peptide. Attached Figure Description
[0010] Figure 1 Conserved sequences shown in different IFN-α subtypes.
[0011] Figure 2 Analysis showing the necessary binding sites of the IFN-α2b complex with IFNAR1-IFNAR2.
[0012] Figure 3 (A) shows the predicted structure of cellular peptide-1, and (B) shows a comparison of similarity with IFN-α2b.
[0013] Figure 4 (A) and (B) show the analytical results of the characteristics of cellular peptide-1.
[0014] Figure 5(A) shows the predicted structure of cellular peptide-2, and (B) shows a comparison of similarity with IFN-α2b.
[0015] Figure 6 (A) and (B) show the analytical results of the characteristics of cellular peptide-2.
[0016] Figure 7 (A) shows the predicted structure of cellular peptide-3, and (B) shows a comparison of similarity with IFN-α2b.
[0017] Figure 8 (A) and (B) show the analytical results of the characteristics of cellular peptide-3.
[0018] Figure 9 (A) shows the predicted structure of cellular peptide-4, and (B) shows a comparison of similarity with IFN-α2b.
[0019] Figure 10 (A) and (B) show the analytical results of the characteristics of cellular peptide-4.
[0020] Figure 11 This displays the cytotoxicity test results for the solvent.
[0021] Figure 12 The results of the cytotoxicity test for Cell Peptide-4 are shown.
[0022] Figure 13 The results of immune function analysis after treatment with cellular peptide-4 for 4 hours are shown.
[0023] Figure 14 The results of immune function analysis after 8 hours of treatment with cellular peptide-4 are shown.
[0024] Figure 15 The results of immune function analysis after 16 hours of treatment with cellular peptide-4 are shown.
[0025] Figure 16 The results of immune function analysis after 24 hours of treatment with cellular peptide-4 are shown. Detailed Implementation
[0026] The present invention provides a composition for regulating an immune response, comprising: a truncated type I IFN, mainly composed of at least one domain selected from helical A, helical B, helical C, helical D, helical E and AB loop; and a buffer, a carrier, and / or an excipient.
[0027] Type I IFNs include, for example, IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω, which can bind to specific cell surface receptor complexes, such as the IFN-α / β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 chains. IFN-α2b belongs to the type I IFN family and has 180 to 190 amino acids. Approximately 20 amino acids from the N-terminus constitute the signal peptide, which is cleaved during secretion, leaving 160 amino acids to form the functional protein that binds to IFNARs on the cell membrane. The structure of type I IFNs consists of five alpha helices: helix A, helix B, helix C, helix D, and helix E. First, helices A, AB loops, and helix E of type I IFNs have relatively high affinity for binding to IFNAR2. Then, they recruit IFNAR1, which has relatively low affinity for type I IFNs, up to helices B, C, and D. Finally, type I IFN, IFNAR1, and IFNAR2 form a ternary complex that is activated via the JAK-STAT signaling pathway to stimulate the expression of specific genes.
[0028] The term "truncated type I IFN" means that, compared to the amino acid sequence or nucleotide of a native type I IFN ("original sequence"), the truncated type I IFN has a shorter amino acid sequence or nucleotide, while retaining at least one functional fragment or domain of the original sequence. That is, one or more sequence fragments are removed from the original sequence of the native type I IFN. In this invention, at least 30% of the length of the original sequence is removed to generate the truncated type I IFN. In one embodiment, at least 40% of the length of the original sequence is removed to generate the truncated type I IFN. In another embodiment, at least 50% of the length of the original sequence is removed to generate the truncated type I IFN.
[0029] In some embodiments, the truncated type I IFN may be simply one or more specific domains of the natural type I IFN, such as helix A, helix B, helix C, helix D, helix E, AB ring, or any combination thereof. In a preferred embodiment, the truncated type I IFN comprises helix A, helix B, helix C, helix D, helix E, AB ring, or any combination thereof. In a preferred embodiment, the truncated type I IFN primarily comprises polypeptides comprising helix B and helix C. In a preferred embodiment, the truncated type I IFN primarily comprises polypeptides comprising helix A and AB ring. In a preferred embodiment, the truncated type I IFN primarily comprises polypeptides comprising helix A, helix B, helix C, and AB ring. In a preferred embodiment, the truncated type I IFN primarily comprises polypeptides comprising helix D and helix E.
[0030] In this invention, the truncated type I IFN can be a peptide having the following amino acid sequence: SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK; SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN; SEQ ID NO. 3: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*, where C* indicates that the amino acid is modified by adding a disulfide bond; SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; and An amino acid sequence having at least 80% identity with any of SEQ ID NO. 1 to 4.
[0031] In this invention, the peptide is designed based on the amino acid sequence of type INF. In the embodiments, the peptide is designed based on the amino acid sequence of INF-α.
[0032] In some embodiments, the truncated type I IFN may have an amino acid sequence that is at least 80% identical to any of SEQ ID NO. 1 to 4. For example, the sequence may have at least 85%, at least 90%, at least 95%, or at least 99% identical to any of SEQ ID NO. 1 to 4.
[0033] In the embodiments, for protein properties such as protein structural stability, signal transduction, protein transport, biocompatibility, etc., the truncated type I IFN peptide may contain additional amino acids in its sequence.
[0034] In some embodiments, the truncated type I IFN peptide may be further modified to improve protein properties, such as affinity for cell membranes and protein structural stability. In some embodiments, the modification may include, but is not limited to, phosphorylation, methylation, glycosylation, ubiquitination, and acetylation.
[0035] In some embodiments, the modification is a lipid modification, such as palmitoylation, myristoylation, or isopentenylation. In some embodiments, the lipid modification, such as palmitoylation, can improve lipophilicity, enhancing the attraction between the modified peptide and the cell membrane, thereby increasing the chance of the modified peptide contacting receptors on the cell membrane. In some embodiments, the lipid modification can allow the modified peptide to cross the cell membrane more efficiently, without being limited by receptor combinations. In a preferred embodiment, the modification is palmitoylation. It is known that the original IFN-α2b protein must bind to two receptors on the cell membrane to act synergistically before entering the cell to exhibit protein activity. However, the receptor requirement for the original IFN-α2b protein can be overcome or reduced by modifying the peptide with palmitic acid, for example, palmitic acid modification can enhance the attraction between the peptide and the cell membrane, thereby increasing the chance of the peptide binding to receptors or facilitating the peptide's passage across the cell membrane.
[0036] The present invention also provides an isolated nucleic acid comprising a nucleotide sequence encoding a peptide consisting primarily of an amino acid sequence selected from the following: SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK; SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN; SEQ ID NO. 3: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*, where C* indicates that the amino acid is modified by adding a disulfide bond; SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; and An amino acid sequence having at least 80% identity with any of SEQ ID NO. 1 to 4.
[0037] In some embodiments, the nucleotide sequence encodes a peptide selected from SEQ ID NO. 1 to 4, or any combination thereof. In some embodiments, the nucleotide sequence may be carried by a vector, which may include, but is not limited to, plasmids, viral vectors, granules, and artificial chromosomes.
[0038] In this invention, the composition can be used to modulate immune responses, for example, responses to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. In one embodiment, the composition can be used to initiate a protective defense mechanism of the immune system to eliminate pathogens or tumors. In one embodiment, the composition can be used to treat and / or prevent viral infections, such as coronavirus infections.
[0039] In this invention, the composition for modulating an immune response may comprise a therapeutically effective dose of a peptide of the truncated type I IFN. In one embodiment, the composition may comprise 1 to several million international units (IU) of the truncated type I IFN. In another embodiment, the composition may comprise 1 to 1,000 micrograms of the truncated type I IFN.
[0040] The composition also includes a buffer, a carrier, and / or an excipient. The buffer, carrier, and / or excipient are pharmaceutically acceptable components, and known buffers, carriers, and excipients may be used, provided they function as buffers, carriers, and excipients in the composition without negatively impacting the peptide. For example, water, acids, alcohols, ethers, sulfoxides, polysaccharides, glycols, stearates, glycerol, and silica may be selected and used in the composition.
[0041] In this invention, the peptide design method may include: performing protein analysis; truncating the original type I IFN sequence based on the analysis results; evaluating the protein properties of the truncated type I IFN sequence; and generating the truncated type I IFN. The protein analysis may include, but is not limited to, analyses of conserved domains, acid dissociation constants, isoelectric point (pI), type I IFN protein characteristics, and binding sites with IFN receptors. The design process is detailed below but is not intended to be limiting. The truncated type I IFN peptide is hereinafter also referred to as a "cytopeptide".
[0042] First, the interaction sites between type I IFN and cell membrane surface receptors are identified using the HOMCOS protein binding prediction program. Second, based on this analysis, candidate cellular peptides are designed by preserving key regions of type I IFN (those suggesting potential influences on biological activity and protein structure). Next, protein analysis software such as Protpi, EnCor, and PROTEIN CALCULATOR v3.4 is used to further calculate the peptide properties, including amino acid type, pI, overall charge at pH 7.40, and charge under different environments (pH 6 to 8). Furthermore, to more closely approximate the structure of the original type I IFN protein, modifications are made to specific positions of the candidate cellular peptide sequence based on secondary structure prediction results, solvent accessibility, and protein binding properties. Finally, the cellular peptide structure is constructed using protein modeling software (such as SWISS-MODEL) based on the established human IFN / IFN receptor complex to confirm the final cellular peptide sequence.
[0043] In this embodiment, the design process is executed to design and select cellular peptides, and these cellular peptides are used for gene expression testing. For example, cells can be treated with different types and dosages of the designed cellular peptides to perform RNA purification and reverse transcription to obtain complementary DNA (cDNA), and the phenotypes of downstream IFN-related pathways such as the JAK-STAT signaling pathway can be detected. By comparing these gene phenotypes, the function of the designed cellular peptides can be evaluated.
[0044] In this invention, the peptide has an amino acid sequence that is truncated from the original IFN-α2b sequence and combines different domains of IFN-α2b. The truncated sequence may be partially similar to the original sequence; however, these sequence alterations simultaneously change the protein structure and may even affect its function. Therefore, from a biological perspective, the truncated sequence is not identical to the original sequence.
[0045] Example Example 1 The following procedure was used to design peptides derived from IFN-α2b.
[0046] Thirteen types of IFN-α were aligned using the ClustalW multiple alignment program in BioEdit software. Figure 1 Conserved sequences are shown across different IFN-α isoforms. The proportion of conserved regions of these amino acid sequences in each α-helix of IFN-α, including helices A, B, C, D, and E, was calculated. (Helix A: 43%, Helix B: 50%, Helix C: 48%, Helix D: 60%, and Helix E: 75%) Next, the proportion of critical binding sites was examined. The inventors in this case listed the critical binding sites between IFN-α2b and IFN α / β receptors based on published literature. Figure 2 Analysis of the key binding sites of the IFN-α2b-IFNAR1-IFNAR2 complex is shown. The IFNAR2 binding sites are marked in yellow, while the IFNAR1 binding sites are marked in blue. Next, the proportion of each α-helical region as a key binding site was calculated: helix A 21%, helix B 30%, helix C 33%, helix D 18%, and helix E 35%.
[0047] Accordingly, the peptides of the present invention are designed to target the conserved region and / or the key binding site, such as helices A, B, C, D, and E. In the following examples, the designed peptides are referred to as cell peptide-1, cell peptide-2, cell peptide-3, and cell peptide-4, respectively.
[0048] Example 2 Cellular peptide-1 has the following amino acid sequence: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK (SEQ ID NO. 1).
[0049] Structural prediction of cellular peptide-1 The structure of cellular peptide-1 was predicted using SWISS-MODEL. A model was constructed in SWISS-MODEL using a template from the PDB database. Figure 3 (A)). Next, the structure of cellular peptide-1 was superimposed with IFN-α2 to compare the similarity between the two. Figure 3 (B)). The prediction results show that cellular peptide-1 consists of two α-helical structures, similar to helix B and helix C of IFN-α2b.
[0050] Characteristics of Cellular Peptide-1 The amino acid composition, pI value, and charge characteristics of cellular peptide-1 were analyzed using the protein calculator program, Protein PI, and Protein CALCULATOR v3.4. The analysis results are as follows: Figure 4 (A) and Figure 4 As shown in (B). Based on the amino acid composition, the hydrophobic / hydrophilic ratio and the basic / acidic amino acid ratio of cellular peptide-1 are close to those of IFN-α2b. Figure 4 (A)). On the other hand, regarding the charge characteristics of cellular peptide-1, it is slightly negatively charged (from -0.3 to -2.5) at pH 6.00 to 8.00, similar to IFN-α2b ( Figure 4 (B)).
[0051] Example 3 Cellular peptide-2 has the following amino acid sequence: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN (SEQ ID NO. 2) Structural prediction of cellular peptide-2 The structure of cellular peptide-2 was predicted using SWISS-MODEL. A model was constructed in SWISS-MODEL using a template from the PDB database. Figure 5 (A)). Next, the structure of cellular peptide-2 was superimposed with IFN-α2 to compare the similarity between the two. Figure 5 (B)). The prediction results show that cellular peptide-2 is similar to the helical A and AB ring structure of IFN-α2b.
[0052] Characteristics of Cellular Peptide-2 The amino acid composition, pI value, and charge characteristics of cellular peptide-2 were analyzed using the protein calculator program, Protein PI, and Protein CALCULATOR v3.4. The analysis results are as follows: Figure 6 (A) and Figure 6 As shown in (B). Based on the amino acid composition, the hydrophobic / hydrophilic ratio and the basic / acidic amino acid ratio of cellular peptide-2 are close to those of IFN-α2b. Figure 6 (A)). On the other hand, regarding the charge characteristics of cellular peptide-2, it is slightly positively charged (from 1.8 to 0.3) at pH 6.00 to 8.00. Figure 6 (B)).
[0053] Example 4 Cellular peptide-3 has the following amino acid sequence: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC* (SEQ ID NO. 3), * indicates that the amino acids in this cellular peptide-3 have been modified by adding disulfide bonds to stabilize the peptide structure.
[0054] Structural prediction of cellular peptide-3 The structure of cellular peptide-3 was predicted using SWISS-MODEL. A model was constructed in SWISS-MODEL using a template from the PDB database. Figure 7 (A)). Next, the structure of cellular peptide-3 was superimposed with IFN-α2 to compare the similarity between the two. Figure 7(B)). The prediction results show that cellular peptide-3 consists of three α-helical structures and a long ring, similar to helix A, helix B, helix C, and AB ring of IFN-α2b.
[0055] Characteristics of Cellular Peptide-3 The amino acid composition, pI value, and charge characteristics of cellular peptide-3 were analyzed using the protein calculator program, Protein PI, and Protein CALCULATOR v3.4. The analysis results are as follows: Figure 8 (A) and Figure 8 As shown in (B). Based on the amino acid composition, the hydrophobic / hydrophilic ratio and the basic / acidic amino acid ratio of cellular peptide-3 are close to those of IFN-α2b. Figure 8 (A)). On the other hand, regarding the charge characteristics of cellular peptide-3, it is slightly negatively charged (from 0.5 to -2.9) at pH 6.00 to 8.00, which is also similar to IFN-α2b ( Figure 8 (B)).
[0056] Example 5 like Figure 1 and Figure 2 As shown, the results indicate that helix E of IFN-α2b is the most conserved region of the IFN-α isoform, and that helix E contains the most critical receptor-binding site. Therefore, cellular peptide-4 was designed targeting helix E.
[0057] The amino acid sequence of cellular peptide-4 was aligned with IFN-α2b using the multiple sequence alignment program Clustal Omega, which was developed by EMBL-EBI (European Molecular Biology Laboratory - European Institute for Bioinformatics). Cellular peptide-4 was derived by truncating the original type I IFN sequence and designing it to target the E region of the helix based on publicly available literature.
[0058] Cellular peptide-4 has the following amino acid sequence: SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE Structural prediction of cellular peptide-4 The structure of cellular peptide-4 was predicted using the protein modeling program SWISS-MODEL based on the established human IFN / IFN receptor complex. First, a model was constructed in SWISS-MODEL using a template from the PDB database. Figure 9 (A)). Furthermore, the structure of cellular peptide-4 was superimposed with IFN-α2 to compare the similarity between the two. Figure 9(B)), green indicates high similarity, while red indicates low similarity. The prediction results show that cellular peptide-4 consists of two α-helical structures, similar to helix D and helix E of IFN-α2b. Figure 9 (A) and (B)).
[0059] Characteristics of cellular peptides First, the interaction site between type I IFN and the receptor on the cell surface was identified using the HOMCOS protein binding prediction software. Second, the cellular peptide was further confirmed using two protein analysis software programs, Port PI and EnCor, to identify peptide characteristics, including amino acid types, amino acid percentages, and pI values. These results confirm the physical properties between the truncated peptide and IFN-α2b. Third, the cellular peptide was evaluated using the online analysis programs CALCULATOR v3.4 and PredictProtein to determine its overall charge at pH 7.40, charge at different environments (pH 6.00 to 8.00), predicted secondary structure, solvent accessibility, and protein binding status.
[0060] The protein analysis program Port pi is used to provide amino acid percentages. The inventors then further calculated the proportions of hydrophobic, hydrophilic, basic, and acidic amino acids in the cellular peptide and IFN-α2b sequence. Additionally, the online analysis program Protein CALCULATOR v3.4 can assist in predicting molecular weight, estimating pI values, and charge.
[0061] The ratio of hydrophobic / hydrophilic and basic / acidic amino acids in this cellular peptide is higher than that of IFN-α2b. On the other hand, regarding the charge characteristics of this cellular peptide, it is mostly positively charged (from 2.1 to 1.2) at pH 6.00 to 8.00, while IFN-α2b is slightly negatively charged (from 0.8 to -3.5) at pH 6.00 to 8.00. In short, the characteristics of this cellular peptide differ from those of full-length IFN-α2b. Figure 10 (A) and (B)).
[0062] Example 6 This incomplete IFN-α structure can still stimulate downstream signaling pathways without the need for IFNAR1. However, compared to the IFN-α-IFNAR1-IFNAR2 ternary complex, the lack of IFNAR1-induced effects may reduce the expression of immune-related genes. To improve the potency of this cellular peptide, the inventors palmitoylated the peptide. Palmitoylation is a lipid modification, chemically synthesized at the N-terminus of the peptide sequence, replacing the cellular modification. Due to the hydrophobic nature of this group, the palmitoylated peptide is more readily accessible to the cell membrane. This cellular peptide can maximize its chances of binding to the extracellular domain of IFNAR2. Subsequently, it can activate the intracellular domain of IFNAR2 to promote the expression of downstream immune-related genes.
[0063] Example 7 The safety of cellular peptide-4 was tested using a cytotoxicity assay.
[0064] Solvent cytotoxicity test Cellular peptide-4 was prepared by chemical synthesis. After several solubility tests, dimethyl sulfoxide (DMSO), which has amphipathic properties, was selected as the solvent for subsequent experiments. HEK293t (human embryonic kidney cells) cells were seeded into 6-well plates, 8 x 10⁸ cells per well. 5 Cells were treated with six different concentrations of DMSO (10%, 5%, 3%, 1%, 0.5%, and 0.25% v / v). The cells were then cultured at 5% CO2 and 37°C for 8 hours (hr). Finally, the cell phenotype was observed using an optical microscope.
[0065] To compare normal and abnormal cell phenotypes, a negative control group not treated with DMSO served as a control. Experimental groups received 10%, 5%, 3%, 1%, 0.5%, and 0.25% v / v DMSO.
[0066] The results showed that various abnormal cell phenotypes, including features of shrinkage and apoptosis, were observed in the 10% v / v and 5% v / v DMSO experimental groups. Figure 11 The 3% v / v DMSO experimental group showed mild apoptosis. Figure 11 The 1% v / v, 0.5% v / v, and 0.25% v / v DMSO experimental groups showed the normal cell phenotype. Figure 11 Therefore, a concentration of less than 1% DMSO is the optimal concentration for use as a solvent.
[0067] Example 8: Cytotoxicity assay of cellular peptide-4 HEK293t cells were seeded into 96-well plates, 2 x 10 cells per well. 4Cells were cultured at 5% CO2 and 37°C. After exposing the cells to four concentrations of cellular peptide-4 (13.7, 41.1, 68.5, and 95.9 μg / ml) for 4 hours, cell viability was assessed by adding Cell Count Kit-8 (CCK-8) reagent (three independent experiments). The cells were cultured for another 2 hours. Finally, the absorbance at 450 nm was measured using a microdisc analyzer.
[0068] DMSO was selected as the solvent for cellular peptide-4. Therefore, four concentrations of DMSO were added as the control group (0.15%, 0.44%, 0.73%, and 1.03% v / v). Ethanol was used as the positive control group. The experimental groups contained 13.7, 41.1, 68.5, and 95.9 μg / ml of cellular peptide-4 combined with 0.15%, 0.44%, 0.73%, and 1.03% v / v DMSO, respectively.
[0069] Data showed that, compared with the DMSO group, three-fold, five-fold, and seven-fold concentrations of cellular peptide-4 did not cause abnormal cell phenotypes or reduce cell viability. Figure 12 ).
[0070] Example 9: Immune Function Test HEK293t cells were seeded into 6-well plates, with 8 x 10 cells per well. 5 Cells were cultured at 5% CO2 and 37°C. Cells in each well were treated with 0.14% v / v DMSO and 13.7 μg / ml cytokinin-4. Cells were then cultured for 4 hours (6 independent experiments). After confirming the cell phenotype, cells were collected using the buffered RLT from the RNeasy mini-kit. Whole RNA was then extracted, and mRNA was converted to cDNA using reverse transcription. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect downstream IFN signaling pathways.
[0071] Untreated cells served as a negative control group, confirming that DMSO treatment did not induce the expression of immune-related genes. This control group consisted of cells treated only with 0.14% v / v DMSO, using DMSO as the solvent for the target cellular peptide. The target genes included JAK1, STAT1, STAT2, and ISG15. The internal control group was GAPDH.
[0072] Clearly, compared with the DMSO control group, the levels of STAT1 and ISG15 were increased after treatment with cellular peptide-4. Figure 13 (Increased by 19% and 18%). These findings confirm that the cellular peptide-4 successfully stimulated the initial regulatory gene STAT1 and subsequent signal transduction to the downstream immune-related gene ISG15.
[0073] 8, 16, and 24-hour processing Perform the same experimental steps, only changing the cell culture time to 8, 16, or 24 hours.
[0074] HEK293t cells were treated with 13.7 μg / ml cellular peptide-4 for 8 hours. Figure 14 ), 16 hours Figure 15 ), and 24 hours ( Figure 16 Compared with the DMSO control group, STAT1 levels remained significantly elevated 8 hours after treatment with cytopeptide-4. This data indicates that the immunomodulatory effect of cytopeptide-4 disappeared after 8 hours of treatment, suggesting that the immunomodulatory effect of cytopeptide-4 does not cause a long-term systemic immune response.
[0075] in conclusion Cellular peptide-4 is not a natural extract; its peptide sequence is modeled after the E-helix region of human INF-α. These tests confirmed that cellular peptide-4 can induce transient regulatory functions of cellular immunity-related genes, superior to traditional interferons. Cellular assays have confirmed that cellular peptide-4 is non-cytotoxic and is expected to significantly reduce the side effects associated with traditional interferon treatment.
[0076] Based on the results of cell experiments, the duration of action is estimated to be 8 to 16 hours. Beyond this estimated duration, it will slowly degrade and decline. Therefore, cellular peptide-4 will not accumulate in the body and pose a safety risk.
[0077] This unique peptide palmitoylation modification design helps improve bioavailability and accelerate cellular absorption.
[0078] In gene expression analysis, cells treated with different time points and concentrations of cellular peptides were collected. Their RNA and reverse-transcribed complementary DNA (cDNA) were purified to detect the expression patterns of downstream IFN-related pathways, such as the JAK-STAT signaling pathway. The expression patterns of genes related to the JAK-STAT pathway in the cellular peptide group were similar to those in the IFN-treated group, showing significant induction compared to the control group. These results indicate that the cellular peptide possesses IFN-like function and can regulate the expression of immune-related genes.
[0079] Experimental data show that this cellular peptide not only possesses interferon-like immune-related gene regulatory functions but also exhibits no cytotoxicity. Therefore, this cellular peptide holds promise for various applications, including as an active pharmaceutical ingredient, a raw material for immunomodulatory products, a non-traditional food ingredient, a health food ingredient, and a skin care product. Notably, this cellular peptide has the potential to replace traditional IFN as a new drug ingredient, opening up tremendous potential for further exploration.
[0080] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.
Claims
1. A composition for regulating an immune response, comprising: The truncated type I interferon (IFN) is mainly composed of at least one domain selected from helical A, helical B, helical C, helical D, helical E, and AB loop; and Buffers, carriers, and / or excipients.
2. The composition of claim 1, wherein, The first type of IFN is IFN-α2b.
3. The composition of claim 1, wherein, This truncated type I IFN is primarily composed of at least one amino acid sequence selected from the following: SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK; SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN; SEQ ID NO. 3: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*, where C* indicates that the amino acid is modified by adding a disulfide bond; SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; and An amino acid sequence having at least 80% identity with any of SEQ ID NO. 1 to 4.
4. The composition of claim 1, wherein, The amino acid sequence was further modified with lipids.
5. The composition of claim 4, wherein, The lipid modification is palmitoylation.
6. The composition of claim 1, wherein, The buffer, carrier, and / or excipient is selected from water, acid, alcohol, ether, sulfoxide, polysaccharide, glycol, stearate, glycerol, and silica.
7. A peptide that regulates an immune response, comprising primarily an amino acid sequence selected from at least one of the following: SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK; SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN; SEQ ID NO. 3: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*, wherein, C* indicates that the amino acid is modified by adding a disulfide bond; SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; and An amino acid sequence having at least 80% identity with any of SEQ ID NO. 1 to 4.
8. The peptide of claim 7, wherein, The peptide was further modified with lipids.
9. The peptide of claim 7, wherein, The lipid modification is palmitoylation.
10. An isolated nucleic acid comprising a nucleotide sequence encoding the peptide of claim 7.
11. A composition for treating or preventing viral infection, comprising: The truncated type INF is primarily composed of at least one structural domain selected from helical A, helical B, helical C, helical D, helical E, and AB loop; and Buffers, carriers, and / or excipients.
12. The composition of claim 11, wherein, The first type of IFN is IFN-α2b.
13. The composition of claim 11, wherein, The truncated type I IFN is primarily composed of at least one amino acid sequence selected from the following: SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK; SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN; SEQ ID NO. 3: C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*, where C* indicates that the amino acid is modified by adding a disulfide bond; SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; and An amino acid sequence having at least 80% identity with any of SEQ ID NO. 1 to 4.
14. The composition of claim 13, wherein, The amino acid sequence was further modified with lipids.
15. The composition of claim 14, wherein, The lipid modification is palmitoylation.
16. The composition of claim 11, wherein, The buffer, carrier, and / or excipient is selected from water, acid, alcohol, ether, sulfoxide, polysaccharide, glycol, stearate, glycerol, and silica.
17. The composition of claim 11, further comprising interferon β and / or interferon γ.
18. The composition of claim 11, further comprising an antiviral agent and / or an anti-inflammatory agent.