Novel peptides and their derivatives capable of stimulating cytokine release

Abstract

The present invention relates to novel peptides capable of stimulating cytokine release and to the use of such peptides as pharmaceutical agents.

Description

[Technical Field]

[0001] The present invention relates to novel peptides capable of stimulating cytokine release and to the use of such peptides as pharmaceutical agents. [Background technology]

[0002] Cytokines play a key role in regulating immune responses. These intercellular messengers, released by innate immune cells, enable a coordinated, potent, and self-limiting response to pathogens and insults. However, over the past two decades, there has been growing interest in the role that cytokines play in cancer immunotherapy.

[0003] Cytokines can directly stimulate immune effector cells and stromal cells at tumor sites, thereby enhancing tumor cell recognition by cytotoxic effector cells. Numerous animal tumor model studies have demonstrated that cytokines have broad antitumor activity, which has translated into numerous cytokine-based approaches for cancer treatment (Lee & Margolin, 2011). Such cytokines exert their antitumor effects directly against the cancer cells or indirectly, for example, through interactions with immune cell populations. Many of these mechanisms remain to be identified, despite promising antitumor effects in a range of animal cancer models.

[0004] For example, IL-2, originally discovered as a "T cell growth factor" to enhance antitumor immune responses by driving T cell proliferation, has since been approved for the treatment of many cancers, including melanoma and renal cell carcinoma (Jiang et al., 2016). In addition to its clinical approval for use in these cancers, the literature also hailed it as a "go-to" cancer immunotherapy, particularly due to its ability to activate the immune system in combination with other anticancer immunotherapies (Jiang et al., 2016). Indeed, IL-2 is considered "the first effective immunotherapy for human cancer" and is likely applicable to all cancer types (Rosenberg, 2014).

[0005] Furthermore, IL-4, originally reported as a "B cell growth factor," enhances antitumor immune responses and directly induces apoptosis in cancer cells, including breast cancer (Nagai and Toi, 2000). Although there is some contradiction regarding the function of IL-4 in tumor immunity, IL-4 has been shown to be the most effective cytokine in inducing immune responses in many cancer prevention and treatment models (Li et al., 2009).

[0006] IL-12 has been considered one of the most promising candidates for tumor immunotherapy in humans because of its ability to activate both the innate (NK cell) and adaptive (cytotoxic T lymphocyte) arms of the immune response (Lasek et al., 2014). IL-12-based therapies have been successful in many types of cancer, including breast cancer, pancreatic cancer, cervical cancer, colorectal cancer, lymphoma, melanoma, multiple myeloma, renal cancer, sarcoma, and liver cancer (Lasek et al., 2014), and many recent clinical trials have confirmed this efficacy (Lasek & Zagozdzon, 2016).

[0007] IFN-γ, also known as type II IFN, can mediate antitumor immunity through various mechanisms, including enhancing immune cell activation and survival, enhancing immune effector function, reducing regulatory T cell immunosuppression, and enhancing cytotoxic function (Parker et al., 2016). Furthermore, IFN-γ has been shown to mediate the inhibition of tumor angiogenesis, a process that drives tumor progression, growth, and metastatic spread in all human cancers (Hayakawa et al., 2002). Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, IL-2, IL-4, IL-12, and IFN-γ are all clearly important cytokines in the development, growth, and spread of human cancers; thus, regulation of these cytokines plays a role in all cancers. Many cytokines are in preclinical development for cancer immunotherapy. However, delivery of therapeutic cytokines is problematic, and researchers are developing alternative strategies, such as recombinant viral vectors for cytokine gene delivery and PEGylation of cytokine proteins to improve host disposition (Lee and Margolin, 2011). Therefore, there is a need to find new methods to enhance the release of these cytokines, including but not limited to IL-2, IL-4, IL-12, and IFN-γ, from host cells and promote anti-tumor immunity. The present invention addresses this challenge. [Means for solving the problem]

[0009] In one aspect of the invention, the amino acid sequence: X1X2X3AX4X5X6X7X8X9X 10 ; (In the formula, X1 is selected from PyroQ and A; X2 is selected from the group consisting of D, E, and A; X3 is selected from the group consisting of T, A, and S; X4 is selected from the group consisting of V, A, I, L, and M; X5 is selected from the group consisting of T and S; X6 is selected from the group consisting of T, A, and S; X7 is selected from the group consisting of H, K, Q and R; X8 is selected from the group consisting of E, A, N, and Q; X9 is selected from the group consisting of D, N, Q, and A; X 10 is selected from the group consisting of N, D, and A or a fragment of a functional variant thereof, An isolated polypeptide comprising:

[0010] In one embodiment, X1 is PyroQ.

[0011] In one embodiment, alternatively, X 1 is A.

[0012] In a second embodiment, X2 is E.

[0013] In a third embodiment, X3 is T.

[0014] In the fourth embodiment, X4 is V.

[0015] In a fifth embodiment, X5 is S.

[0016] In the sixth embodiment, X6 is S.

[0017] In a seventh embodiment, X7 is H.

[0018] In an eighth embodiment, X8 is E.

[0019] In a ninth embodiment, X9 is Q.

[0020] In the tenth embodiment, X 10 is D.

[0021] In one embodiment, an isolated polypeptide is provided comprising a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 47, or a functional variant thereof.

[0022] In another aspect of the present invention, an isolated polynucleotide is provided, wherein the isolated polynucleotide is: (a) an isolated polynucleotide encoding a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 47, or a functional variant thereof, as identified above; (b) a nucleotide sequence complementary to (a); Includes:

[0023] In a further aspect of the present invention, there is provided a nucleic acid construct comprising at least one nucleic acid sequence encoding at least one polypeptide as defined in any of SEQ ID NOs: 1 to 47, or a functional variant or homologue thereof, wherein at least one of said sequences is preferably operably linked to a regulatory sequence, which in one embodiment is a constitutive or strong promoter.

[0024] In another aspect of the invention, there is provided a vector comprising at least one of the above polynucleotides.

[0025] In a further aspect of the invention, there is provided a host cell comprising at least one nucleic acid construct.

[0026] In another aspect of the invention, there is provided an isolated polypeptide, polynucleotide, nucleic acid construct, or host cell described herein for use as a medicament.

[0027] In a further aspect of the present invention, there is provided a method of treatment comprising administering to an individual or patient in need thereof at least one isolated polypeptide, polynucleotide, or nucleic acid construct described herein.

[0028] In another aspect of the invention, there is provided at least one isolated polypeptide, polynucleotide, nucleic acid construct, or host cell as described herein for use in the treatment of cancer.

[0029] In a further aspect of the present invention, there is provided a method of treating cancer comprising administering at least one isolated polypeptide, polynucleotide, nucleic acid construct, or host cell described herein to an individual or patient in need thereof.

[0030] In one example, the cancer may be selected from one of the following: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of the blood tissue, glioma, lymphoma, etc. In one embodiment, the cancer is liver cancer.

[0031] In another aspect of the present invention, there is provided a pharmaceutical composition comprising at least one isolated polypeptide, polynucleotide, nucleic acid construct or host cell described herein and a pharmaceutically acceptable carrier.

[0032] In a further aspect of the invention, there is provided a method of increasing cytokine levels, the method comprising administering to a target cell or a patient at least one isolated polypeptide, polynucleotide, nucleic acid construct or host cell as described herein.

[0033] In a final aspect of the invention, there is provided the use of at least one isolated polypeptide, polynucleotide, nucleic acid construct or host cell to enhance or stimulate cytokine release.

[0034] In one embodiment, the cytokine is at least one of IL-2, IL-4, IL-12, and IFN-γ.

[0035] The invention is further described in the following non-limiting drawings. [Brief explanation of the drawings]

[0036] [Figure 1A] FIG. 1 shows that the polypeptide of the present invention is not toxic to HepG2 (human liver cancer cell line) cell proliferation when administered at different concentrations. [Figure 1B]FIG. 1 shows that the polypeptide of the present invention is not toxic to HepG2 (human liver cancer cell line) cell proliferation when administered at different concentrations. [Figure 2A] FIG. 1 shows the effect of administration of polypeptides on the secretion of cytokines (IL-2, IL-4, IFN-γ, and IL-12) from mouse splenic lymphocytes. [Figure 2B] FIG. 1 shows the effect of administration of polypeptides on the secretion of cytokines (IL-2, IL-4, IFN-γ, and IL-12) from mouse splenic lymphocytes. [Figure 2C] FIG. 1 shows the effect of administration of polypeptides on the secretion of cytokines (IL-2, IL-4, IFN-γ, and IL-12) from mouse splenic lymphocytes. [Figure 2D] FIG. 1 shows the effect of administration of polypeptides on the secretion of cytokines (IL-2, IL-4, IFN-γ, and IL-12) from mouse splenic lymphocytes. [Figure 2E] FIG. 1 shows the effect of administration of polypeptides on the secretion of cytokines (IL-2, IL-4, IFN-γ, and IL-12) from mouse splenic lymphocytes. [Figure 3] FIG. 1 shows the effect of administration of polypeptide-treated mouse spleen lymphocyte serum (containing cytokines) on HepG2 cell proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention is further described below. In the following sections, different aspects of the invention are specified in more detail. Each specified aspect can be combined with any other aspect unless otherwise specified. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length joined together by peptide bonds.

[0038] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutant, or synthetic DNA or RNA molecules, and analogs of DNA or RNA produced using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode an mRNA or protein product. The term also includes genes. The terms "gene" or "gene sequence" are used broadly to refer to a DNA nucleic acid associated with a biological function, i.e., a gene may include introns and exons, as in a genomic sequence, or may include only the coding sequence, as in a cDNA, and / or may include cDNA in combination with regulatory sequences.

[0039] The inventors have identified a number of peptides corresponding to modified fragments of rabbit α-1-antiproteinase. Fragments of the nucleotide sequence ... X1X2X3AX4X5X6X7X8X9X 10 ; (In the formula, X1 is selected from PyroQ and A; X2 is selected from the group consisting of D, E, and A; X3 is selected from the group consisting of T, A, and S; X4 is selected from the group consisting of V, A, I, L, and M; X5 is selected from the group consisting of T and S; X6 is selected from the group consisting of T, A, and S; X7 is selected from the group consisting of H, K, Q and R; X8 is selected from the group consisting of E, A, N, and Q; X9 is selected from the group consisting of D, N, Q, and A; X 10is selected from the group consisting of N, D, and A The consensus sequence was:

[0040] It can enhance or stimulate the release of cytokines from innate immune cells.

[0041] In one embodiment of the invention, the following amino acid sequence: X1X2X3AX4X5X6X7X8X9X 10 ; (In the formula, X1 is selected from PyroQ and A; X2 is selected from the group consisting of D, E, and A; X3 is selected from the group consisting of T, A, and S; X4 is selected from the group consisting of V, A, I, L, and M; X5 is selected from the group consisting of T and S; X6 is selected from the group consisting of T, A, and S; X7 is selected from the group consisting of H, K, Q and R; X8 is selected from the group consisting of E, A, N, and Q; X9 is selected from the group consisting of D, N, Q, and A; X 10 is selected from the group consisting of N, D, and A An isolated polypeptide comprising:

[0042] In one embodiment X2 is E. In a second embodiment, X3 is T. In a third embodiment, X4 is V. In a fourth embodiment, X5 is S. In a fifth embodiment, X6 is S. In a sixth embodiment, X7 is H. In a seventh embodiment, X8 is E. In an eighth embodiment, X9 is Q. In a ninth embodiment, X 10 is D.

[0043] In a particularly preferred embodiment, the peptide comprises the sequence PyroQETAVSSHEQD (SEQ ID NO: 1 or SEQ ID NO: 25) or a functional variant thereof. This peptide is sometimes referred to herein as Peptide 1.

[0044] In another embodiment, the peptide comprises or consists of a sequence selected from any one of SEQ ID NOs: 2-24 or SEQ ID NOs: 26-47.

[0045] PyroQ, as referred to herein, is also known as pyroglutamine. The structure of PyroQ is as follows:

[0046] [ka] For the avoidance of doubt, PyroQ is also known as PyroE, PyroGln, PyroGlu or pyroglutamate, and the terms may be used interchangeably herein, i.e., PyroQ and PyroE are structurally identical.

[0047] The structure of PyroQ when bound to the N-terminus of a peptide is shown below, where the wavy line indicates the point of attachment:

[0048] [ka] PyroQ can exist as either the (R) or (S) enantiomer as follows:

[0049] [ka] In another aspect, the present invention provides an isolated polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 to 47, or a fragment thereof, or a functional variant thereof.

[0050] The term "variant" or "functional variant" as used herein with respect to any of SEQ ID NOS: 1-47 refers to a variant sequence or portion thereof that retains the biological function of the intact, non-variant sequence. Functional variants also include variants with sequence changes that do not affect function, e.g., in non-conserved residues. Also included are variants that are substantially identical, i.e., have only minor sequence variations and are biologically active. Nucleic acid or amino acid sequence changes that result in the production of a different amino acid at a given site without affecting the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the hydrophobic amino acid alanine can be substituted with a codon encoding another, less hydrophobic residue, e.g., glycine, or a more hydrophobic residue, e.g., valine, leucine, or isoleucine. Similarly, changes resulting in the substitution of another negatively charged residue, such as aspartic acid for glutamic acid, or another positively charged residue, such as lysine for arginine, would also be expected to produce a functionally equivalent product. Each of the above suggested modifications is within the ordinary skill in the art, as is determination of retention of biological activity of the encoded products.

[0051] As used in any aspect of the invention described herein, a "variant" or "functional variant" refers to a variant that has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

[0052] Two nucleic acid sequences or polypeptides are said to be "identical" if the sequences of nucleotides or amino acid residues in the two sequences are identical, respectively, when aligned for maximum correspondence, as described below. The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical, or have a specified percentage of identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence over a comparison window, as determined using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. When percentage sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), thereby not altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, one sequence usually serves as a reference sequence and is compared with the test sequence.When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the sequence identity ratio of the test sequence to the reference sequence based on the program parameters.Non-limiting examples of algorithms suitable for determining sequence identity and sequence similarity ratios are BLAST and BLAST 2.0 algorithms.

[0053] The polypeptides of the invention may comprise additional amino acids, such as N-terminal additions useful for purifying the polypeptide, for example, binding tags and cleavage recognition sites.

[0054] In one example, the binding tag binds to glutathione, and the tag can be glutathione S-transferase (GST). In another example, the tag can be biotin. The binding tag target is preferably immobilized on a solid support, allowing the bound polypeptide to be easily isolated from unbound products. Other suitable binding tags immobilized on similar solid supports can be used.

[0055] In other examples, the cleavage recognition site comprises a sequence that recognizes thrombin, enterokinase, or factor Xa, among others. Preferably, this site is internal to or adjacent to the binding tag.

[0056] Polypeptides of the present invention may be modified, including but not limited to, any post-translational modification such as glycosylation, alkylation (e.g., methylation), acetylation, amidation, hydroxylation, ubiquitination, sulfation, and phosphorylation, any chemical modification, or any modification, including non-covalent and covalent attachment to other proteins or peptides.

[0057] A further aspect of the present invention provides a method for producing or purifying the polypeptide, comprising expressing in a host cell a vector comprising a nucleotide sequence encoding any one of SEQ ID NOS: 1-47, wherein the vector preferably comprises a regulatory sequence as described herein, and more preferably comprises a nucleotide sequence encoding a binding tag; allowing the expressed polypeptide to bind to a target of the binding tag; and releasing the bound polypeptide from the target. The host cell may be a eukaryote, such as a mammal, other vertebrate or invertebrate, insect, fungus, or plant cell; or a prokaryote, such as a bacterium; and vectors derived from bacteria, yeast, other eukaryotes, other non-eukaryotes, or viral sequences may be used. The method may then comprise cleaving the polypeptide at the recognition site.

[0058] Polypeptides can be produced or synthesized by any method known to those of skill in the art, for example, in one embodiment, the polypeptides are produced using chemical synthesis. An example of a method for synthesizing a polypeptide of the present invention is provided in Example 2.

[0059] In another aspect of the present invention, an isolated polynucleotide is provided, wherein the isolated polynucleotide is: (a) a nucleotide sequence encoding a polypeptide selected from any one of SEQ ID NOs: 1 to 47, or a variant or fragment thereof; or (b) a nucleotide sequence complementary to (a); (c) a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (b); or (d) a nucleic acid sequence encoding a polypeptide of any one of SEQ ID NOs: 1 to 47, which is capable of hybridizing to any one of the nucleic acid sequences of (a) to (c) under stringent conditions as defined herein; Includes:

[0060] Hybridization of the sequence can be performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" are intended to mean conditions under which a probe hybridizes to its target sequence detectably more strongly than other sequences (e.g., at least twice as strongly as background). Stringent conditions depend on the base sequence and vary depending on the circumstances. By controlling the stringency of the hybridization and / or washing conditions, it is possible to identify a target sequence that is 100% complementary to the probe (homologous probe). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequence so that less similar sequences are detected (heterologous probe). Generally, the probe length is less than about 1000 nucleotides, preferably less than 500 nucleotides.

[0061] Typically, stringent conditions would involve a salt concentration of less than about 1.5M Na ion, typically about 0.01-1.0M Na ion (or other salt) at a pH of 7.0-8.3, and a temperature of at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Hybridization times are generally less than about 24 hours, typically about 4-12 hours. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.

[0062] In a further aspect of the present invention, there is provided a nucleic acid construct or vector comprising a nucleic acid sequence encoding a polypeptide selected from any one of SEQ ID NOS: 1-47 or a functional variant or homolog thereof, wherein the sequence is preferably operably linked to a regulatory sequence. The regulatory sequence can be any type of promoter, such as a constitutive, strong, regulated, or inducible promoter, that results in expression of the nucleic acid when expressed in a target or host cell. Examples include, but are not limited to, viral promoters such as the cytomegalovirus (CMV) promoter and simian vacuolating virus 40 (SV40), as well as non-viral promoters such as elongation factor (EF)-1 and actin.

[0063] The term "promoter" refers to a nucleic acid control sequence, typically located upstream of the transcription initiation site of a gene, that is responsible for binding RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Included within the term are transcriptional regulatory sequences derived from classical eukaryotic genomic genes (including a TATA box, with or without a CCAAT box sequence, necessary for accurate transcription initiation) and additional regulatory elements (i.e., upstream activating sequences, enhancers, and silencers) that alter gene expression in response to developmental and / or external stimuli or in a tissue-specific manner. The term also includes transcriptional regulatory sequences from classical prokaryotic genes, which may include a -35 box sequence and / or a -10 box transcriptional regulatory sequence.

[0064] As used herein, the term "operably linked" refers to a functional linkage between a promoter sequence and a gene of interest such that the promoter sequence is capable of initiating transcription of the gene of interest.

[0065] Another aspect of the present invention provides a vector comprising the polynucleotide described above. The vector may be a bacterial or yeast plasmid, cosmid, bacteriophage, artificial chromosome, or plant or mammalian virus. Preferably, the vector is an expression vector, also known as an expression construct. In one embodiment, an expression vector may contain an origin or replication, at least one selectable marker, and a multiple cloning site suitable for insertion of a nucleic acid sequence to be expressed. Expression vectors may be generated by any one of a number of techniques known to those skilled in the art.

[0066] In a further aspect of the present invention, there is provided a host cell comprising a nucleic acid construct. The host cell may be prokaryotic or eukaryotic, and may include bacterial cells, fungal cells such as yeast, plant cells, insect cells, or mammalian cells. By way of example only, the mammalian host cell may be selected from CHO (Chinese Hamster Ovary) cells, COS, HEK, or HeLa. Alternatively, the host cell may be a lymphocyte (B lymphocyte or T lymphocyte), a macrophage or mast cell, or an immune cell such as a liver or spleen cell, an endothelial cell, a fibroblast, or a stromal cell. Also provided is a host cell comprising an exogenous polynucleotide according to the above aspect of the present invention. Preferably, the host cell expresses the polynucleotide.

[0067] In another aspect of the invention, there is provided a method of producing a polypeptide as described herein, comprising introducing into a host cell and expressing a nucleic acid construct as described, and isolating the polypeptide.

[0068] In another aspect of the present invention, there is provided a method for producing a polypeptide as described herein, comprising introducing and expressing a nucleic acid construct as described in a host cell, and isolating the polypeptide. The nucleic acid construct is introduced into the host cell via a process known as transformation or transfection. The terms "introduction," "transformation," or "transfection" as used herein encompass the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for introduction. The polynucleotide may be transiently or stably introduced into the host cell and may remain non-integrated, for example as a plasmid. Alternatively, it may be integrated into the host genome.

[0069] Transformation is now a routine technique for many species. Advantageously, any of several transformation methods can be used to introduce the gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that enhance the uptake of free DNA, direct injection of DNA into host cells, particle bombardment, viral or pollen-mediated transformation, and microprojection.

[0070] In another aspect of the present invention, there is provided a method of increasing cytokine levels, comprising administering to a target cell or a patient in need thereof at least one or any combination of the isolated polypeptides, polynucleotides, or nucleic acid constructs described herein.

[0071] In one embodiment, the cytokine is at least one of IL-2, IL-4, IL-12, and IFN-γ.

[0072] In a further embodiment, cytokine release is increased by 10-200%, more preferably 10-150%, compared to levels in control cells. In one embodiment, the control cells are unstimulated (i.e., not administered with the peptide).

[0073] In one embodiment, the level of IL-2 is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150% or more compared to the level in a control. In a specific embodiment, the peptide is SEQ ID NO: 1 or SEQ ID NO: 25, and the level of enhancement is 5-40%, more preferably 5-15%.

[0074] In another embodiment, IL-4 levels are increased by 5-100%, more preferably at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to control levels. In a specific embodiment, the peptide is SEQ ID NO: 1 or SEQ ID NO: 25, and the level of enhancement is 5-40%, more preferably 10-20%, compared to control levels.

[0075] In further embodiments, the level of IFN-γ is increased by 2-100%, more preferably at least 2%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to the level in a control. In a specific embodiment, the peptide is SEQ ID NO: 1 or SEQ ID NO: 25, and the level of increase is 25-60%, more preferably 30-40%, compared to the level in a control.

[0076] In other embodiments, IL-12 levels are increased by 10-400%, more preferably at least 10%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400% or more compared to levels in a control. In a specific embodiment, the peptide is SEQ ID NO: 1 or SEQ ID NO: 25, and the increased level is 150-250%, more preferably 180-220%, compared to levels in a control.

[0077] In another aspect of the present invention there is provided at least one or any combination of the isolated polypeptides or polynucleotides described herein for use as a medicament.

[0078] In a further aspect of the present invention, there is provided a method of treatment comprising administering to an individual or patient in need thereof at least one or any combination of the isolated polypeptides, polynucleotides, or nucleic acid constructs described herein.

[0079] In another aspect of the present invention there is provided at least one or any combination of the isolated polypeptides, polynucleotides or nucleic acid constructs described herein for use in the treatment of cancer.

[0080] In a further aspect of the present invention, there is provided a method of treating cancer, the method comprising administering to a patient in need thereof at least one or any combination of the isolated polypeptides, polynucleotides, or nucleic acid constructs described herein.

[0081] In another aspect of the present invention, there is provided the use of at least one or any combination of the isolated polypeptides, polynucleotides, or nucleic acid constructs described herein in the preparation of a medicament for the treatment of cancer.

[0082] In one example, the cancer may be selected from one of the following: pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of the blood tissue, glioma, lymphoma, etc. In one embodiment, the cancer is liver cancer.

[0083] Another aspect of the present invention provides a method for reducing tumor cell proliferation, comprising administering at least one isolated polypeptide, polynucleotide, or nucleic acid construct to a target cell or patient in need thereof. In one embodiment, the level of reduction is 10-60%, more preferably at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more, compared to control levels. In a specific embodiment, the peptide is SEQ ID NO: 1 or SEQ ID NO: 25, and the level of reduction is 5-20%, more preferably 10-15%, compared to control levels.

[0084] As used herein, a control can be an individual, patient or cell that has not been treated with at least one polypeptide of the present invention.

[0085] In another aspect of the present invention, there is provided a pharmaceutical composition comprising any one or at least one of the peptides, polynucleotides, vectors or constructs described herein and a pharmaceutically acceptable carrier. The compositions will typically be formulated using well-known methods prior to administration to a patient.

[0086] The administration of the polypeptide or pharmaceutical composition of the present invention can be carried out orally or parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal, mucosal, or intranasal administration. In addition to the active ingredient, such compositions may contain a suitable pharmaceutically acceptable carrier, including excipients and other ingredients that facilitate processing of the active compound into a preparation.

[0087] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration, which allow the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for ingestion by a subject.

[0088] Pharmaceutical preparations for oral use can be prepared by combining the active compound with a solid excipient, optionally grinding the mixture, and processing the granular mixture, after adding suitable additional compounds if desired, to obtain tablets or dragee cores. Suitable excipients include carbohydrate or protein fillers such as sugars including lactose, sucrose, mannitol, and sorbitol; starches derived from corn, wheat, rice, potato, or other plants; celluloses such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; gums such as gum arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof.

[0089] Dragee cores can be provided with suitable coatings such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and concentrated sugar solutions which may contain suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound.

[0090] Orally administrable formulations include push-fit capsules made of gelatin and soft sealed capsules made of gelatin, as well as tablet coatings such as glycerol or sorbitol. Push-fit capsules can contain the active ingredients mixed with fillers or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0091] Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compound. For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilizers or agents to increase the solubility of the compound and allow for the preparation of highly concentrated solutions. The pharmaceutical composition may also contain adjuvants to enhance or modulate antigenicity.

[0092] For topical or intranasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation.

[0093] In one embodiment, the polypeptides, polynucleotides or nucleic acid constructs can be used as cellular adjuvants or immunotherapies to mediate innate or adaptive immunity, chemoattract cells, and enhance antigenicity. In one embodiment, the polypeptides, polynucleotides or nucleic acid constructs of the invention are co-administered with antigens or other immunotherapies.

[0094] While the above disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including how to make and use the invention, and the best mode thereof, the following examples will enable one of ordinary skill in the art to practice the invention and are provided to provide a complete written description thereof. However, those skilled in the art will appreciate that the details of the examples should not be construed as limiting the invention, the scope of which should be understood from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0095] "And / or," when used herein, should be considered a specific disclosure of each of the two specified features or components. For example, "A and / or B" should be considered a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0096] Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0097] The above applications, and all literature and sequence accession numbers cited therein or pending therein ("Cited Literature"), as well as all literature cited or referenced in the Cited Literature, and all literature cited or referenced herein ("Cited Literature"), and all literature cited or referenced herein, as well as manufacturer's instructions, descriptions, product specifications, and product sheets for any products cited herein, are hereby incorporated by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0098] The invention is described in the following non-limiting examples. [Example]

[0099] Experimental Overview The inventors evaluated the antitumor activity of peptides by immune system enhancement using the following three separate experiments: 1. Evaluate the ability to inhibit the growth of human tumor cells.

[0100] 2. The ability of treated mouse splenic lymphocytes to enhance the secretion of immune cell-related cytokines.

[0101] 3. Finally, serum from mouse splenic lymphocytes after peptide treatment was isolated and added directly to human tumor cells to evaluate the inhibitory effect on tumor cell growth.

[0102] experiment 1 - Cytotoxicity of peptides against tumor cells a) Cell culture - HepG2 cells were grown in DMEM high glucose medium (containing 10% FBS by volume) and cultured at 37°C in a 5% CO2 incubator. Trypsin digestion and regular cell passage were performed. b) CCK-8 detection of cell proliferation HepG2 cells in the logarithmic growth phase were prepared as a cell suspension and 5 × 10 3 Cells were seeded into each well of a 96-well flat-bottom plate. After overnight incubation, cells were grown in adherent culture and replaced with medium containing different concentrations of polypeptide dissolved in PBS / DMSO. Blank solvent was used as a negative control. Each sample was performed in triplicate and incubated for 15 hours. Then, cck-8 (10 μl / well) was added and incubated for 1 hour. The absorbance A at 450 nm was measured using an enzyme-linked immunosorbent assay. The results of this experiment are shown in Figure 1. As shown in Figure 1, the peptide itself does not affect tumor cell proliferation and does not exhibit cytotoxicity.

[0103] 2. Study on immunological activity of candidate peptides Mouse splenic lymphocytes were prepared and tested for immune activity with candidate peptides in stimulation experiments. a) Male Kunming (KM) mice were killed by cervical dislocation, and the spleens were aseptically dissected. The spleens were then placed in a dish containing 5 mL of lymphocyte isolation solution and crushed. Cells were collected by centrifugation at 800 g for 30 minutes, and the supernatant was removed. The cells were washed with 10 mL of RPMI 1640 cell culture medium (1640) and isolated by centrifugation at 250 g for 10 minutes. The cells were resuspended in 1640 and fetal bovine serum (FBS), and the lymphocyte concentration was adjusted to 5 × 10 6The concentration of cells was adjusted to 1 / mL. 100 μL of suspended cells were added to each well of a 96-well plate, and different concentrations of candidate polypeptides (dissolved in either PBS or DMSO) were added in triplicate and incubated for 24 hours. ConA / LPS was used as a positive control. b) ELISA - After incubation, the samples were centrifuged, and the supernatants were separated and tested according to the instructions of the ELISA kit for cytokine concentration detection. Briefly, 50 μL of RD-14 was added to each well, followed by 50 μL of sample (standard, control, and supernatant isolated from step A). ​​After mixing, the solution was incubated at room temperature for 2 hours. The samples were washed five times, and 100 μL of binding solution was added and incubated at room temperature for 2 hours. The samples were then washed five times, and 100 μL of substrate solution was added. After incubation at room temperature in the dark for 30 minutes, 100 mL of quenching solution was added, and the following OD values ​​(450 nm) were measured. The results of this experiment are shown in Figure 2. As shown in Figure 2, as the concentration of the polypeptide increased, the amounts of IL-2, IL-4, IFN-γ, and IL-12 each increased.

[0104] 3. Immunoreactivity of candidate peptides against tumor cells Splenic lymphocytes were stimulated according to (2a), and supernatants containing immune cytokines were obtained by centrifugation. The supernatants were added to overnight-cultured HepG2 cells and further cultured for 24 hours. Two negative controls were performed using (1) blank solvent and (2) polypeptides dissolved in PBS / DMSO. Cell viability was detected by MTT assay, and their effects on tumor cell proliferation were analyzed. The results of this experiment are shown in Figure 3. As shown in Figure 1, the concentration "0.00" represents splenic supernatants not challenged with peptides, and "(1.00)" refers to peptides administered directly to HepG2 cells at a concentration of 1 mg / mL, thus containing no cytokines (as in Experiment 1, shown in Figure 1). As shown in Figure 3, splenic supernatants challenged with various concentrations of the claimed peptides significantly reduced cell proliferation in a model human hepatoma cell line (HepG2 cells). [Example]

[0105] Exemplary Methods for Producing Peptides In one example, the peptides of the present invention can be produced using the following method: a) 200 mg of 2-chlorotrityl resin (1.0 mmol / g) was loaded into a manual peptide reactor, swollen with DCM for 30 minutes, and then dried. The corresponding Fmoc-protected amino acid (0.4 mmol, 2 equiv.), DIEA (0.4 mmol, 2 equiv.), and appropriate amounts of DMF and DCM were mixed by bubbling N2 for 1 hour. Then, MeOH (1.0 mmol, 5 equiv.) and DIEA (0.4 mmol, 2 equiv.) were added and reacted for 30 minutes to cap any unreacted sites. b) The resin was washed with DMF (x5) and then reacted with 20% piperidine in DMF for 10 min to remove the Fmoc group. 20% piperidine in DMF was added twice. The resulting resin was then washed with DMF and confirmed by the chloranil test. c) The following Fmoc amino acid (0.4 mmol, 2 equiv.), HBTU (0.4 mmol, 2 equiv.), and DIEA (0.4 mmol, 2 equiv.) in DMF / DCM were added to the resin, mixed by bubbling N2 for 1 hour, and the resin was checked by chloranil test. If the coupling was incomplete, another portion of the Fmoc amino acid and coupling reagent was added and allowed to react for an additional hour. d) Repeat steps (b) and (c) until the array is complete. e) After the removal of the last Fmoc group, the resin is transferred to another glass vial, and a cleavage cocktail (95% TFA, 2% EDT, 2% TIS, and 1% water) is added at room temperature and left for 3 hours. The resulting mixture is filtered, concentrated, and transferred to cold diethyl ether (10x concentrated mixture). The peptide is isolated by centrifugation. The pellet is then dried under reduced pressure and then purified by HPLC.

[0106] [Table 1]

[0107] [Table 2]

[0108]

Table 3

[0109]

Table 4

Claims

1. An isolated polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 24, wherein the structure of PyroQ in the amino acid sequence is as follows: 【Chemical 1】 An isolated polypeptide represented by:

2. A nucleic acid construct comprising a polynucleotide encoding at least one isolated polypeptide of claim 1.

3. A vector comprising the nucleic acid construct of claim 2.

4. A host cell comprising at least one vector according to claim 3.

5. 5. An isolated polypeptide, nucleic acid construct, vector or host cell according to any one of claims 1 to 4 for use as a medicament.

6. 5. The isolated polypeptide, nucleic acid construct, vector or host cell according to any one of claims 1 to 4 for use in the treatment of cancer.

7. A pharmaceutical composition for treating cancer, comprising at least one of the isolated polypeptide, nucleic acid construct, vector or host cell according to any one of claims 1 to 4.

8. 8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier.

9. 8. The pharmaceutical composition of claim 7, for increasing the level of or stimulating the release of a cytokine by administration to a target cell or a patient.

10. 10. The pharmaceutical composition according to claim 9, wherein the cytokine is at least one of IL-2, IL-4, IFN-γ, and IL-12.

Images