Galectin-targeted immunotherapy

Abstract

The present disclosure provides virus-like particles comprising viral structural proteins and galectin epitope peptides, as well as compositions or vaccines comprising them, and their uses in medicine, particularly in immunotherapy.

Description

[Technical field]

[0001] The present disclosure relates to galectin-targeted immunotherapy. [Background technology]

[0002] Galectins are members of the lectin family that show high affinity for β-galactosides. In mammals, about 15 galectins have been discovered that are encoded by LGALS genes and are numbered consecutively. Currently, only galectins-1, -2, -3, -4, -7, -7B, -8, -9, -9B, -9C, -10, -12, -13, -14 and -16 have been identified in humans.

[0003] Galectin-1 (Gal-1) is a member of a highly conserved family of animal lectins that bind to N-acetyllactosamine (LacNAc; Galβ1-4GlcNAc)-containing glycoproteins or glycolipids (Barondes SH 1994). Gal-1 is significantly upregulated and secreted in many tumors. High levels of Gal-1 are associated with tumor aggressiveness, metastasis, and poor survival in many cancers (Astorgues-Xerri L 2014). Previous studies have illustrated that Gal-1 has emerged as a novel regulatory checkpoint that promotes immune evasion programs by inducing T cell exhaustion, limiting T cell survival, favoring the proliferation of regulatory T cells, inactivating natural killer cells, and polarizing myeloid cells toward an immunosuppressive phenotype (Banh A 2011). Galectin-1 expression is upregulated by hypoxia, and glycosylation-dependent galectin-receptor interactions control angiogenesis (Rabinovich 2014) (Xu-Yun Zhao 2010). Thus, targeting galectin-glycan interactions may hinder tumor progression by simultaneously enhancing antitumor immunity and suppressing aberrant angiogenesis (Thijssen VL 2006).

[0004] Due to the essential and multifunctional role of galectins and / or galectin-binding glycoconjugates in the process of cancer development and prognosis, various candidate inhibitors that may block the interaction between these molecules have been proposed as potential anticancer drugs (Non-Patent Document 7: Lucile Astorgues-Xerri 2014). Currently, the best-known galectin antagonists are glycomimetics, derivatives or analogs of galactosides, which target the canonical carbohydrate-binding site of galectins (Non-Patent Document 8: Koonce NA 2017). Peptide and antibody Gal-1 inhibitors have also been reported for their ability to antagonize galectin-receptor interactions (Non-Patent Document 9: Diego O. Croci 2012, Non-Patent Document 10: van der Schaft DW 2002).

[0005] Galectin-3 (Gal-3) is a member of a highly conserved family of animal lectins that bind to β-galactoside-containing glycoconjugates (glycoproteins or glycolipids) (Henderson MA NC 2006; Mourad-Zeidan AA 2008). Gal-3 is unique among other galectin family proteins in its structure, which consists of two domains: a carboxyl-terminal domain that contains the carbohydrate-binding region and an amino-terminal domain that consists mainly of tandem repeats of nine amino acids that bridge carbohydrate and non-carbohydrate ligands (Figure 12A) (Liu 1990; Barondes SH 1994). Gal-3 is secreted by various types of cells, including monocytes, macrophages, and epithelial cells, but mainly by macrophages (Reynolds 2005; MacKinnon AC 2008). Secreted extracellular Gal-3 forms homodimers or pentamers, which are important for biological functions. It has been demonstrated that the N-terminal domain of galectin-3 is important for the formation of protein oligomers (Figure 12B) (Non-Patent Document 13: Liu 1990, Non-Patent Document 16: Dumic J 2006, Non-Patent Document 17: Kuklinski S 1998, Non-Patent Document 18: Lepur A 2012). The released protein can function as an extracellular molecule to activate cells, mediate cell-cell and cell-ECM interactions, induce migration of various types of cells, and negatively regulate T cell receptor signaling (Non-Patent Document 19: Yang RY 1996). Gal-3 has been shown to be increased in various models of fibrotic diseases and patients, including pulmonary fibrosis, liver fibrosis, systemic sclerosis, and myocardial fibrosis (Non-Patent Document 20: MA Henderson NC 2008, Non-Patent Document 21: Barman, et al. 2019, Non-Patent Document 22: Nishi Y 2007, Non-Patent Document 23: De Boer 2010). This suggests that Gal-3 may be an important mediator of tissue fibrosis and an effective therapeutic target for tissue fibrosis.To date, preclinical and clinical studies of investigational galectin-3 inhibitors have shown protective effects against fibrotic disorders (Non-Patent Document 24: Nikhil Hirani 2017, Non-Patent Document 25: Yu L 2013).

[0006] One of the major success stories in medicine over the past 100-150 years has been vaccines targeting various infectious diseases. Vaccines, together with antibiotics, have likely been more important to human health than any other part of the human body. The success of vaccines has led to increased interest in using vaccine technology to treat non-infectious diseases such as allergy, autoimmunity, and cancer. However, the targets of these diseases are generally self-antigens, which can cause problems with efficacy. Compared to non-self-antigen bacterial, viral, or parasitic proteins, it is considerably more difficult to induce strong antibody responses against self-antigens due to resistance mechanisms (Hellman 2008; Falk Saupe 2015).

[0007] Alphaviruses comprise a series of genetically, structurally, and serologically related mosquito-borne viruses in the family Togaviridae. Alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki forest virus, Middleburg virus, Chikungunya virus (CHIKV), O'nyong-nyong virus, Ross River virus, Barmah forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kiziragachi virus, Highland J virus, Fort Morgan virus, Nudum virus, and Boggy Creek virus. The capsid, a structural subunit containing a single viral protein, associates with the RNA genome in an icosahedral nucleocapsid. In virions, the capsid is surrounded by a lipid envelope that is covered with a regular array of transmembrane protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2.

[0008] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of standard native viruses but lack the viral genome, and thus may provide safer and cheaper vaccine candidates. A handful of VLP-based prophylactic vaccines are currently commercially available worldwide: GlaxoSmithKline's Engerix® (Hepatitis B virus) and Cervarix® (Human Papillomavirus), and Merck and Co., Inc.'s Recombivax HB® (Hepatitis B virus) and Gardasil® (Human Papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical evaluation, such as influenza virus, parvovirus, Norwalk virus, and various chimeric VLPs. Despite their success in preclinical trials, many others are still limited to small-scale platform studies. The implications of large-scale VLP production are discussed in relation to process control, monitoring, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly. Successful breakthroughs of VLP-based vaccines are briefly presented along with the latest results of clinical trials and recent developments in chimeric VLP-based technologies directed towards either therapeutic or prophylactic vaccination.

[0009] To date, VLP-based vaccines have been produced against over 30 different viruses that infect humans and other animals, including AAV (adeno-associated virus), H5N3 (avian influenza), BFDV (budgie fledgling disease virus), BTV (bluetongue virus), Ebola, Enterovirus 71, GHPV (goose hemorrhagic polyomavirus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis delta virus), HEV (hepatitis E virus), HIV, HPV (human papilloma virus), IBDV (infectious bursal disease virus), influenza A, influenza A H1N1, influenza A, influenza B ... Examples of such viruses include H3N2, JC polyomavirus, Marburg, MS2, IPCV (Indian peanut cramp virus), NDV (Newcastle disease virus), No (Norovirus), Nv (Norwalk virus), PhMV (ground cherry mottle virus), polyomavirus, PPV (porcine parvovirus), RHDV (rabbit hemorrhagic disease virus), rotavirus, SARS, SIV (simian immunodeficiency virus), SV40 (simian virus 40), SVDV (swine vesicular disease virus), etc. (Non-Patent Document 28: Expert Rev. Vaccines 9(10), 1149-1176, 2010).

[0010] US Patent No. 9,353,353 (Patent Document 1) discloses a virus-like particle (VLP) containing one or more viral structural proteins of Chikungunya virus, which is useful for formulating a vaccine or antigenic composition against Chikungunya that induces immunity against the infection or at least one symptom thereof. US Patent No. 9,487,563 (Patent Document 2) discloses modified virus-like particles (VLPs) of alphaviruses or flaviviruses and a method for enhancing the production of modified VLPs for use in preventing or treating alphavirus and flavivirus-mediated diseases. US Patent No. 9,249,191 (Patent Document 3) discloses virus-like particles of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), in which the virus-like particle contains at least one antigen inserted into the E2 envelope protein to form a fusion protein. U.S. Patent No. 9,969,986 discloses an alphavirus virus-like particle, which comprises alphavirus viral structural proteins including envelope protein E3, which has been modified to contain at least one foreign antigen inserted into its furin cleavage site. The cited references are incorporated herein by reference. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 9,353,353 [Patent Document 2] U.S. Patent No. 9,487,563 [Patent Document 3] U.S. Patent No. 9,249,191 [Non-patent literature]

[0012] [Non-Patent Document 1] [ PMC free article ] [ PubMed ] Barondes SH, Cooper DN, Gitt MA, Leffler H. 1994. Galectins. Structure and function of a large family of animal lectins. J Biol Chem 269:20807–20810.

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[0013] [Means for solving the problem]

[0014] The present disclosure relates to galectin-targeted immunotherapy.

[0015] In a first aspect, the present disclosure provides a virus-like particle comprising a viral structural protein and at least one galectin antigen.

[0016] In a second aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the virus-like particle provided in the first aspect of the application.

[0017] In a third aspect, the disclosure provides a vector comprising the nucleic acid molecule of the second aspect, and optionally an expression control sequence operably linked to the nucleic acid molecule.

[0018] In a fourth aspect, the present disclosure provides a pharmaceutical or vaccine composition comprising: (a) a virus-like particle of the first aspect, a nucleic acid molecule of the second aspect, and / or a vector of the third aspect; and (b) a pharma- ceutically acceptable carrier.

[0019] In a fifth aspect, the present disclosure provides a galectin-targeted immunotherapy comprising administering an effective amount of the virus-like particle of the first aspect, the nucleic acid molecule of the second aspect, and / or the vector of the third aspect to a subject in need thereof.The method can be useful for treating or preventing cancer, inflammatory disease, and fibrotic disease.Methods are also provided for modulating immune response in a subject, immunostimulating a subject, producing antibodies against galectin in a non-human mammal; modulating immune response in a subject; immunostimulating in a subject; inhibiting galectin-glycan interaction in a subject; or inhibiting galectin activity.

[0020] In a sixth aspect, the present disclosure relates to the use of a virus-like particle of the first aspect, a nucleic acid molecule of the second aspect, and / or a vector of the third aspect, comprising: For modulating an immune response in a subject, for immunostimulating a subject, for producing antibodies against galectins in a non-human mammal; for modulating an immune response in a subject; for immunostimulation in a subject; for inhibiting galectin-glycan interactions in a subject; The present invention provides a use for the manufacture of a pharmaceutical composition or kit for immunotherapy.

[0021] In a sixth aspect, the present disclosure relates to the use of a virus-like particle of the first aspect, a nucleic acid molecule of the second aspect, and / or a vector of the third aspect, comprising: for treating or preventing cancer or inflammatory diseases; for producing antibodies against galectins in mammals; for modulating immune responses; for immunostimulation; for inhibiting galectin-glycan interactions; or for inhibiting galectin activity. There is provided a use for the manufacture of a pharmaceutical or vaccine composition of the fourth aspect for immunotherapy. [Brief description of the drawings]

[0022] [Figure 1]Preparation of VEEV Gal-1 VLPs. From left to right, each lane is a VEEV VLP carrying Gal-1 epitope peptide #5, #7, #12, and #14, respectively. The rightmost lane is a control VLP without antigenic peptide. [Diagram 2] [Figure 2A] Immunogenicity of CHIKV-VLPs carrying Gal-1 epitope peptides #5 to #10 (derived from human) in naive BALB / c mice. [Figure 2B] Immunogenicity of CHIKV-VLPs carrying Gal-1 epitope peptides #5 (derived from human) and #12 (derived from mouse) in naive BALB / c mice. [Diagram 3] [Figure 3A] Expression of VEEV-VLPs carrying Gal-1 epitope peptides. [Figure 3B] 3D structure of Gal-1 protein. Gal-1 epitope peptides #5 and #12 encode the four β-sheets S3, S4, S5, and S6 near the carbohydrate recognition domain (CRD). [Figure 3C] 3D structure of Gal-1 protein. Gal-1 peptides #7 and #14 encode the β-sheets F4 and F5 that may be important in homophilic dimerization. [Figure 3D] Anti-human Gal-1 antibody titers in the serum of mice immunized with VEEV-VLPs carrying Gal-1 epitope peptides #5, #7, #12, and #14. [Figure 3E] Anti-mouse Gal-1 antibody titers in the serum of mice immunized with VEEV-VLPs carrying Gal-1 epitope peptides #5, #7, #12, and #14. [Figure 3F] Comparison between Gal-1 epitope peptides derived from human Gal-1 and mouse Gal-1. Amino acids that differ between the epitope peptides are underlined. [Figure 4] [Figure 4A] Anti-mouse Gal-1 antibody titers in the serum of mice immunized with CHIKV-VLPs carrying Gal-1 epitope peptides #7 and #12. [Figure 4B] Antibody titers against CHIKV spike protein in the serum of mice immunized with VEEV-VLPs carrying Gal-1 epitope peptides #7 and #12. [Figure 4C] Immunoglobulin subclasses of antibodies in the serum of mice immunized with CHIKV-VLPs carrying Gal-1 epitope peptides #12 and #14. [Diagram 5] [Figure 5A] Effect of CHIKV-VLPs carrying Gal-1 epitope peptides #7 and #12 on Lewis lung carcinoma in mice. (Control) [Figure 5B] Effect of CHIKV-VLPs carrying Gal-1 epitope peptides #7 and #12 on Lewis lung carcinoma in mice. (Control VLP) [Figure 5C] Effect of CHIKV-VLPs carrying Gal-1 epitope peptides #7 and #12 on Lewis lung carcinoma in mice. (Gal-1 #7 VLP) [Figure 5D] Effect of CHIKV-VLPs carrying Gal-1 epitope peptides #7 and #12 on Lewis lung carcinoma in mice. (Gal-1 #12 VLP) [Figure 5E] Tumor weight at endpoint in each group. There was no statistically significant difference in tumor volume between the control group and the VLP-treated group (Figures 5A-D) and (Figure 5E), but tumor growth in mice treated with Gal-1 #12 was slower than that in the other groups. [Figure 6] [Figure 6A] The effect of CHIKV-VLP carrying Gal-1 epitope peptides #12 and #14 on mouse HPV+ head and neck squamous cell carcinoma cell model. The tumor growth of mice treated with Gal-1 #14 VLP vaccine was slower than that of control, but the difference between any groups was not statistically significant. [Figure 6B] The effect of CHIKV-VLP carrying Gal-1 epitope peptides #12 and #14 on the endpoint weight of mouse HPV+ head and neck squamous cell carcinoma cell model. [Figure 7] Molecular size and purity of VEEV-VLPs carrying Gal-3 epitope peptides #17, #21, #22 and #23 and control VLPs. [Figure 8] Immunogenicity of VEEV-VLPs carrying Gal-3 epitope peptides #17, #21, #22 and #23 in mice. [Figure 9] Flow cytometry analysis. Sera from mice immunized with VLPs carrying Gal-3 epitope peptides #17 and #22 were analyzed for binding to cell surface Gal-3. [Figure 10]Neutralizing ability of anti-Gal-3 antibodies induced in mice by VLPs carrying Gal-3 epitope peptides #17 and #22. [Figure 11] [Figure 11A] Effect of VLPs carrying Gal-3 epitope peptides #22 and #23 on the mouse STAM nonalcoholic steatohepatitis (NASH) model. (Steatosis score) [Figure 11B] Effect of VLPs carrying Gal-3 epitope peptides #22 and #23 on the mouse STAM nonalcoholic steatohepatitis (NASH) model. (Inflammation score) [Figure 11C] Effect of VLPs carrying Gal-3 epitope peptides #22 and #23 on the mouse STAM nonalcoholic steatohepatitis (NASH) model. [Balloning score] [Figure 12] (A) The structure of galectin-3 protein consists of the N-terminal domain, which has a 12 amino acid N-terminal region containing the serine six (6) phosphorylation site and a 100 aa long collagen-like repeat sequence. The carbohydrate recognition domain (CDR) 130 aa includes the C-terminus and contains the NWGR motif; (B) The pentameric structure of galectin-3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] definition By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0024] As used herein, the term "adjuvant" refers to a compound that, when used in combination with a particular immunogen in a formulation, will enhance, modify or alter the resulting immune response. In certain embodiments, adjuvants are used in combination with VLPs. In other embodiments, adjuvants are used in combination with DNA vaccines. Modification of immune response includes enhancing or expanding the specificity of either or both antibody and cellular immune responses. Modification of immune response can also mean reducing or suppressing a particular antigen-specific immune response.

[0025] As used herein, "ameliorate" means to lessen, suppress, attenuate, alleviate, arrest or stabilize the occurrence or progression of a disease or its symptoms.

[0026] As used herein, "alteration" refers to a change in an amino acid or nucleotide at a specific position with respect to a polypeptide or polynucleotide sequence. As used herein, alteration includes substitution, deletion, or insertion of an amino acid or nucleotide at a specific position of a polypeptide or polynucleotide. In some embodiments, an alteration of an alphavirus capsid protein nuclear localization signal includes substitution of a charged amino acid (e.g., lysine or arginine) with an uncharged amino acid (e.g., alanine or asparagine, or any amino acid except basic charged amino acids such as lysine or arginine).

[0027] As used herein, "analog" refers to a molecule that is not identical but has similar functional or structural properties. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide, but has certain biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide. Such biochemical modifications may, for example, increase the protease resistance, membrane permeability, or half-life of the analog without altering ligand binding. An analog may also include unnatural amino acids.

[0028] As used herein, "comprises," "comprising," "containing," "having," and the like, may have the meaning ascribed to them in U.S. patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meaning ascribed to them in U.S. patent law, but the terms are open-ended, thus permitting the presence of more than what is described, but excluding prior art embodiments, so long as the basic or novel characteristics of what is described are not altered by the presence of more than what is described.

[0029] As used herein, "detect" means to identify the presence, absence or amount of the analyte to be detected.

[0030] As used herein, "disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0031] As used herein, "effective amount" refers to the amount of agent required to ameliorate the symptoms of disease compared to untreated patients. The effective amount of active compound used to practice the present invention for the prevention or treatment of disease varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0032] As used herein, a "fragment" refers to a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0033] As used herein, an "isolated polynucleotide" refers to a nucleic acid molecule (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which the nucleic acid molecule of the present disclosure is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; that is incorporated into an autonomously replicating plasmid or virus; that is incorporated into the genomic DNA of a prokaryote or eukaryote; or that exists as a molecule separated independently from other sequences (e.g., cDNA, or fragments of a genome or cDNA produced by PCR or restriction enzyme digestion). In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0034] As used herein, an "isolated polypeptide" refers to a polypeptide of the present disclosure separated from components that naturally accompany the polypeptide. Typically, a polypeptide is isolated when it is at least 60% by weight free from proteins and naturally occurring organic molecules that naturally accompany the polypeptide. Preferably, a preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the present disclosure. An isolated polypeptide of the present disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0035] As used herein, "marker" means any protein or polynucleotide that has an alteration in expression levels or activity that is associated with a disease or disorder.

[0036] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise acquiring the agent.

[0037] As used herein, "reduce" means a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0038] As used herein, "reference" means a standard or control condition.

[0039] As used herein, "reference sequence" refers to a defined sequence that is used as a standard for sequence comparison.A reference sequence may be a subset or the entirety of a particular sequence; for example, it may be a segment of a full-length cDNA sequence or gene sequence, or it may be a complete cDNA sequence or gene sequence.For polypeptides, the length of a reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.For nucleic acids, the length of a reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer number therebetween or thereabout.

[0040] As used herein, "specifically binds" means a compound or antibody that recognizes and binds to a polypeptide or protein, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide or protein.

[0041] As used herein, "subject" means a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0042] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, or 50.

[0043] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or its associated symptoms. Although not intended to preclude, it will be understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0044] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is understood to be inclusive.

[0045] Unless specifically stated otherwise or clear from the context, as used herein, the terms "a," "an," and "the" are understood to be in the singular or in the plural.

[0046] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the range normally accepted in the art, for example, within 2 standard deviations of the mean value. About can be understood as within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.1%, within 0.05%, or within 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term about.

[0047] Detailed Description of the Invention The present disclosure provides a virus-like particle comprising a viral structural protein and a galectin antigen.

[0048] As used herein, the term "galectin antigen" refers to any antigenic structure derived from any of the galectin proteins that can be recognized by the immune system and / or stimulate a cellular immune response and / or stimulate the production of antigen-specific antibodies. The galectin epitope peptide may be a fragment of a naturally occurring galectin protein, or a fragment of a naturally occurring galectin protein with some modifications. The naturally occurring galectin protein may be Galectin-1 (Gal-1) or Galectin-3 (Gal-3). The galectin protein is preferably a human galectin protein, particularly preferably a human Galectin-1 protein or a human Galectin-3 protein. In one embodiment, the modified fragment has at least 80%, 85%, 90%, 95%, or 98% amino acid sequence identity with a fragment of a naturally occurring galectin protein. In one embodiment, the modified peptide fragment is a mutant in which at most 10% of the amino acids are deleted, substituted, and / or added to a fragment of a naturally occurring galectin protein.

[0049] The amino acid sequences of naturally occurring human galectin-1 and human galectin-3 are as follows: Galectin-1 [Homo sapiens] MACGLVASNLNLKPGECLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGDANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVCITFDQANLTVKLPDGYEFKFPNRLNLEAINYMAADGDFKIKCVAFD (SEQ ID NO: 7)

[0050] Galectin-3 [human] (GenBank accession number: AAB86584.1) MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (SEQ ID NO: 8)

[0051] Examples of galectin antigens, i.e., galectin epitope peptides, include the following: Gal-1 epitope peptide #5: SFVLNLGKDSNNL S LHFNPRFNAHGDANTIV S NSKDGGAWGTEQREAVFPFQPGS (SEQ ID NO:9) #7: ANLTVKLPDGYEFKFPNRLNLEA (SEQ ID NO: 10) #12: SFVLNLGKDSNNL S LHFNPRFNAHGDANTIV S NTKEDGTWGTEHREPAFPFQPGS (SEQ ID NO: 11) #14: ADLTIKLPDGHEFKFPNRLNMEA (SEQ ID NO: 12)

[0052] Gal-3 epitope peptide #17: ADNFSLHDALSGSGNPNPQGWPGAWGNQPA (SEQ ID NO: 13) #21: YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGA (SEQ ID NO: 14) #22: YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGA (SEQ ID NO: 15) #23: ADSFSLNDALAGSGNPNPQGYPGAWGNQPA (SEQ ID NO: 16)

[0053] Gal-1 epitope peptide #5 is a fragment from human Gal-1, #12 is the corresponding fragment from mouse Gal-1 where the cysteine ​​in the wild-type sequence is replaced by a serine (underlined), Gal-1 epitope peptide #7 is a fragment from wild-type human Gal-1, and #14 is the corresponding fragment from mouse wild-type Gal-1.

[0054] Gal-3 epitope peptide #17 consists of a human N-terminal Gal-3 epitope peptide. Gal-3 epitope peptides #21 and 22 consist of human Gal-3 N-terminal repeat epitope peptides, with #22 having a longer repeat sequence than that of #21. #23 encodes a mouse Gal-3 N-terminal peptide.

[0055] In this disclosure, "alphavirus" refers to an RNA-containing virus that belongs to the Togaviridae family of viruses. Exemplary Togaviridae viruses include, but are not limited to, Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki Forest virus, Middleburg virus, Chikungunya virus (CHIKV), O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kiziragati virus, Highland J virus, Fort Morgan virus, Ndum virus, Boggy Creek virus, and Okelbo virus.

[0056] In the virus-like particles provided by the present application, the viral structural protein and the antigen can be linked through at least one first attachment site present on the viral structural protein and at least one second attachment site present on the antigen.

[0057] As used herein, a "first attachment site" and a "second attachment site" each refer to a site at which two or more substances are linked to one another.

[0058] The viral structural protein and the antigen may be directly or indirectly fused, in one embodiment, one or two linkers may be interposed between the N-terminal residue of the antigen and the viral structural protein and / or between the C-terminal residue of the antigen and the viral structural protein.

[0059] The antigen or viral structural protein can be cut and replaced by a short linker. In some embodiments, the antigen or viral structural protein comprises one or more peptide linkers. Typically, the linker is composed of 2 to 25 amino acids (e.g., 2, 3, 4, 5 or 6 amino acids). Usually, the linker is 2 to 15 amino acids long, but in certain circumstances, the linker may be only one, such as a single glycine (G) residue.

[0060] In one embodiment, a nucleic acid molecule in which a polynucleotide encoding a viral structural protein is genetically fused to a polynucleotide encoding an antigen is expressed in a host cell (e.g., a mammalian cell (e.g., 293F cell) such that the first and second attachment sites are linked through a peptide bond. In this case, the viral structural protein and the antigen are linked through a peptide bond. For this embodiment, the first and / or second attachment sites can be genetically modified from the original protein or antigen. For example, the first attachment site is modified from the viral structural protein such that the protein is conjugated to the antigen through a linker peptide, including SG, GS, SGG, GGS, and SGSG. When the viral structural protein is chemically conjugated to the antigen, the first and second attachment sites can be linked through a chemical cross-linker. Examples of cross-linkers include, but are not limited to, SMPH, sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, SIA and other cross-linkers available from Pierce Chemical Company.

[0061] Preferred examples of alphaviruses include chikungunya virus and Venezuelan equine encephalitis virus. Examples of chikungunya virus may include chikungunya virus strain 37997 and OPY-1. Examples of Venezuelan equine encephalitis virus may include Venezuelan equine encephalitis virus strain TC-83.

[0062] The viral structural protein may include capsid protein and / or envelope protein, fragments thereof or complexes thereof. Thus, the viral structural protein used in the present application may include capsid protein and / or envelope protein and / or fragments or derivatives thereof. In one embodiment, the virus-like particle provided by the present disclosure includes capsid, E3, E2 and E1 proteins, and a galectin antigen.

[0063] Preferably, the antigen may be linked to a viral structural protein of Chikungunya virus or a viral structural protein of Venezuelan Equine Encephalitis virus as a genetically engineered fusion protein.

[0064] Under physiological conditions, E3 can be dissociated from E2 after furin cleavage. In one embodiment, the furin cleavage site located in E3 can be mutated to prevent the furin site from being cleaved. For example, an antigen can be inserted into the furin cleavage site to introduce a mutation into the furin cleavage site. In this embodiment, the virus-like particle provided can consist of or include capsid, E3, E2, and E1 proteins, where E3 binds to E2 to form a single protein, and antigen is inserted into the E3 region. For example, the virus-like particle provided by the present application can be formed by assembling 240 capsids, 240 E1 proteins, 240 proteins, each of which has E2 bound to E3 and antigen inserted into each of the E3 regions.

[0065] The viral structural protein of chikungunya virus or the viral structural protein of Venezuelan equine encephalitis virus used in this application may be a naturally occurring viral structural protein or a modified protein thereof. The modified protein may be a fragment of a naturally occurring viral structural protein. In one embodiment, the modified protein has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity with a naturally occurring viral capsid and / or envelope protein. In one embodiment, the modified protein is a mutant in which at most 10% of amino acids are deleted, substituted and / or added to the naturally occurring viral capsid and / or envelope protein. For example, a K64A or K64N mutation can be introduced into the capsid of the viral structural protein of Venezuelan equine encephalitis virus used in this application.

[0066] An exemplary viral structural protein sequence of a Chikungunya virus is provided under Genbank Accession No. ABX40006.1 (SEQ ID NO: 1). Another exemplary viral structural protein sequence of a Chikungunya virus is provided under Genbank Accession No. ABX40011.1 (SEQ ID NO: 2).

[0067] An exemplary Venezuelan Equine Encephalitis virus viral structural protein is shown in SEQ ID NO:3.

[0068] In one embodiment, the first attachment site may comprise an amino group, preferably the amino group of a lysine residue, and the second attachment site may comprise a sulfhydryl group, preferably the sulfhydryl group of a cysteine.

[0069] In one embodiment, a chikungunya virus-like particle or Venezuelan equine encephalitis virus-like particle can be provided, comprising a chikungunya virus or Venezuelan equine encephalitis virus structural protein and at least one galectin antigen, wherein the at least one galectin antigen is inserted into E3 of the viral structural protein, and the chikungunya virus structural protein or Venezuelan equine encephalitis virus structural protein and the galectin antigen are expressed as a fusion protein. The galectin antigen may be directly or indirectly inserted into E3 of the viral structural protein.

[0070] For the viral structural proteins of Chikungunya virus, at least one galectin antigen can be inserted in place of the furin cleavage site (RKRR) from 322R to 325R of E3 of SEQ ID NO: 1 or 2, or the site corresponding to the furin site (RKRR) from 322R to 325R of SEQ ID NO: 1 or 2. For example, for the viral structural proteins of Chikungunya virus, at least one galectin antigen is inserted between residue H at position 321 and residue S at position 326 of SEQ ID NO: 1 or 2; between P at position 320 and S at position 326 of SEQ ID NO: 1 or 2; or between S at position 319 and S at position 326 of SEQ ID NO: 1 or 2, or the site corresponding thereto. The VLP_CHI 0.56 vector can be used to prepare Chikungunya virus-like particles in which a galectin antigen is inserted between residue 321 and residue 326 of SEQ ID NO: 1 or 2. When a galectin antigen is inserted between residues 321 and 326 of SEQ ID NO: 1 or 2, the virus-like particle provided by the present application can be a Chikungunya virus-like particle comprising an E2 and E3 complex, a capsid and E1, in which case at least one galectin antigen is inserted into the E3 region.

[0071] For the viral structural proteins of Venezuelan equine encephalitis virus, at least one galectin antigen can be inserted in place of the furin cleavage site (RKRR) from 331R to 334R of E3 of SEQ ID NO: 3, or a site equivalent to the furin cleavage site from 331R to 334R of SEQ ID NO: 3. For example, for the viral structural proteins of Venezuelan equine encephalitis virus, at least one galectin antigen is inserted between G at position 330 and S at position 335 of SEQ ID NO: 3; between P at position 329 and S at position 335 of SEQ ID NO: 3; or between C at position 328 and S at position 335 of SEQ ID NO: 3, or a site equivalent thereto. The VLP_VEEV 0.66 vector can be used to prepare Venezuelan equine encephalitis virus-like particles in which an antigen is inserted between residues 330 and 335 of SEQ ID NO: 3. When an antigen is inserted between residues 330 and 335 of SEQ ID NO:3, the virus-like particle provided by the present application can be a Venezuelan Equine Encephalitis virus-like particle comprising an E2 and E3 complex, a capsid and E1, where at least one galectin antigen is inserted in the E3 region.

[0072] As used herein with respect to amino acid positions of two proteins or amino acid sequences, the term "corresponding to" is used to indicate that when the two proteins are subjected to a standard sequence alignment (e.g., using the BLASTp program), the amino acid residue at a particular amino acid position of the first sequence corresponds or matches that amino acid position of the second protein. Thus, amino acid position "A" of the first protein will correspond to amino acid position "B" of the second sequence if residues at positions "A" and "B" correspond to each other in a sequence alignment.

[0073] The viral structural proteins of Chikungunya virus and Venezuelan equine encephalitis include capsid, E1, E2, 6K, and E3. 6K is naturally cleaved during the assembly process and removed from the VLP. In this specification and claims, the term "viral structural protein" refers not only to those having 6K, but also to those after 6K has been removed.

[0074] The 6K sequences of CHIKV and VEEV used in the working example are as follows: CHIKV OPY-1 strain, 6K: 749-809aa of SEQ ID NO: 1 atyqeaaiylwneqqplfwlqaliplaalivlcnclrllpcccktlaflavmsvgahtvsa (SEQ ID NO: 4) CHIKV strain 37997, 6K: 749-809aa of SEQ ID NO:2 atyyeaaaylwneqqplfwlqaliplaalivlcnclkllpcccktlaflavmsigahtvsa (SEQ ID NO: 5) VEEV TC-83 strain, 6K: 758-813aa of SEQ ID NO:3 ettwesldhlwnnnqqmfwiqlliplaalivvtrllrcvccvvpflvmagaagaga (SEQ ID NO:6)

[0075] The nucleic acid molecules provided herein include any nucleic acid molecule that encodes a polypeptide or fragment thereof of the present disclosure. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will usually exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule that encodes a polypeptide or fragment thereof of the present disclosure. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will usually exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence can usually hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to a pairing that forms a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or between portions of the complementary polynucleotide sequences under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0076] For example, stringent salt concentrations will usually be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will usually include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as necessary, various levels of stringency are achieved. In a preferred embodiment, hybridization will be performed in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization will be performed in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization will be performed in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be readily apparent to one of skill in the art.

[0077] In most applications, the washing steps following hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, washing stringency can be increased by lowering salt concentration or by increasing temperature. For example, stringent salt concentrations for washing steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps will usually include a temperature of at least about 25°C, more preferably at least about 42°C, even more preferably at least about 68°C. In a preferred embodiment, washing steps will be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washing steps will be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will be performed at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

[0078] Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0079] Sequence identity is usually measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions usually include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity can use the BLAST program, where closely related sequences are indicated by a probability score between e<”3” and e<”100”.

[0080] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0081] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0082] In some embodiments of the present disclosure, the protein may contain mutations that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded protein or the way in which the protein is made. Nucleotide variants may be created for a variety of reasons, such as to optimize codon expression for a particular host, see U.S. Patent Application Publication No. 2005 / 0118191, which is incorporated herein by reference in its entirety for all purposes.

[0083] In addition, the nucleotides can be sequenced to ensure that the correct coding region has been cloned and does not contain any unwanted mutations. The nucleotides can be subcloned into an expression vector (e.g., baculovirus) for expression in any cell. Those skilled in the art will appreciate that a variety of subcloning methods are available and possible.

[0084] Isolated nucleic acid can be, but need not be, substantially purified.For example, a nucleic acid that is isolated in a cloning vector or an expression vector is not pure in that it may only comprise a small percentage of the material in the cell in which it resides.Such a nucleic acid is isolated as the term is used herein because it can be easily manipulated by standard techniques known to those skilled in the art.

[0085] Expression of Polypeptides Generally, VLPs comprising an alphavirus viral structural protein and at least one gelactin antigen can be produced by transforming a suitable host cell with all or a portion of a nucleic acid molecule encoding the polypeptide or a fragment thereof in a suitable expression vehicle.

[0086] Those skilled in the art of molecular biology will appreciate that any of a wide variety of expression systems can be used to provide recombinant proteins. The exact host cell used is not critical to the present disclosure. The polypeptides of the present disclosure can be produced in prokaryotic hosts (e.g., E. coli) or eukaryotic hosts (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells). Such cells are available from a wide variety of sources (e.g., American Type Culture Collection, Rockland, MD; see also, e.g., Ausubel et al., supra). Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells are S. cerevisiae, Kluyveromyces lactis (K. lactis), Candida species including C. albicans and C. glabrata, Aspergillus nidulans, Shizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero cells and Hep-2 cells. Xenopus laevis ovary cells or other cells of amphibian origin can also be used.Prokaryotic host cells include bacterial cells, such as E. coli, B. subtilis, and Mycobacterium.

[0087] Methods for cloning said proteins are known in the art. For example, genes encoding viral structural proteins of certain alphaviruses, such as CHIKV or VEEV, can be isolated by RT-PCR from polyadenylated mRNA extracted from cells infected with said viruses. The resulting product genes can be cloned into vectors as DNA inserts. The term "vector" refers to a vehicle by which nucleic acids can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which can replicate autonomously or integrate into the chromosome of a host cell. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, poly-lysine conjugated DNA or RNA, peptide conjugated DNA or RNA, liposome conjugated DNA, etc., but which do not replicate autonomously. In many, but not all, common embodiments, the vectors of the invention are plasmids or bacmids.

[0088] The present disclosure further provides nucleotides encoding proteins, including chimeric molecules, cloned into expression vectors capable of expression in cells resulting in the formation of VLPs. An "expression vector" is a vector, such as a plasmid, that is capable of promoting the expression as well as replication of a nucleic acid incorporated therein.

[0089] Typically, the nucleic acid molecule to be expressed is "operably linked" to a promoter and / or enhancer and is under the transcriptional regulatory control of the promoter and / or enhancer.

[0090] There are various expression systems for producing the polypeptide of the present disclosure.Expression vectors useful for producing such polypeptides include, but are not limited to, chromosomal, episomal and viral vectors, such as bacterial plasmid-derived, bacteriophage-derived, transposon-derived, yeast episomal-derived, insertion element-derived, yeast chromosomal element-derived, baculovirus, papovavirus, such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus-derived vectors, and combinations thereof.

[0091] The constructs and / or vectors provided herein include alphavirus polynucleotides encoding structural proteins, including envelope or capsid proteins or portions thereof described herein. The vectors can be, for example, phage, plasmid, viral, or retroviral vectors. The constructs and / or vectors containing the nucleotides are required to be operably linked to a suitable promoter, such as, but not limited to, the CMV promoter, the phage lambda PL promoter, the E. coli lac, phoA, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs. Other suitable promoters are known to those skilled in the art depending on the host cell and / or the desired expression rate. The expression construct further contains sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcript expressed by the construct preferably includes a translation initiation codon at the beginning of the polypeptide to be translated and a termination codon appropriately arranged at the end of the polypeptide to be translated.

[0092] It will be preferred that the expression vector contains at least one selection marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Preferred among the vectors are viral vectors such as baculovirus, poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canine adenovirus), herpes virus and retrovirus. Other vectors that can be used with the present disclosure include vectors for use in bacteria, including pQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among the preferred eukaryotic vectors are pFastBacl, pWINEO, pSV2CAT, pOG44, pXTl and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be readily apparent to those skilled in the art.

[0093] Recombinant constructs can be prepared and used to transfect, infect or transform eukaryotic and / or prokaryotic cells and to express viral proteins, including those described herein. Thus, the disclosure provides a host cell that contains a nucleic acid encoding the viral structural genes of an alphavirus, including capsid, E3, E2, 6K, and El, or portions thereof, and / or any of the chimeric molecules described above, and that comprises a vector (or vectors) that permits expression of the viral structural genes of an alphavirus, including capsid, E3, E2, 6K, and El, or portions thereof, and / or any of the chimeric molecules described above, in said host cell under conditions that permit formation of a VLP.

[0094] In one embodiment, the vector is a recombinant baculovirus. In another embodiment, the recombinant baculovirus is transfected into an insect cell. In a preferred embodiment, the cell is an insect cell. In another embodiment, the insect cell is an Sf9 cell.

[0095] In another embodiment, the vector and / or host cell comprises nucleotides encoding alphavirus genes, including capsid, E3, E2, 6K, and E1, or portions thereof, as described.

[0096] In another embodiment, the vector and / or host cell consists essentially of the alphavirus capsid, E3, E2, 6K, and E1, or portions thereof, as described herein. In a further embodiment, the vector and / or host cell consists of the alphavirus proteins including the capsid, E3, E2, 6K, and E1, or portions thereof, as described herein. These vectors and / or host cells contain the alphavirus core, E3, E2, 6K, and E1, or portions thereof, as described herein, and may contain additional cellular components such as cellulVLProteins, baculovirus proteins, lipids, carbohydrates, etc.

[0097] One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, Wis.). According to this expression system, the DNA encoding the polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of T7 regulatory signals, expression of the polypeptide is achieved by inducing expression of T7 RNA polymerase in the host cell. This is usually achieved using a host strain that expresses T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant polypeptide is then isolated according to standard methods known in the art, for example, the methods described herein.

[0098] Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system uses a GST gene fusion system designed to express genes or gene fragments as fusion proteins at high levels, allowing rapid purification and recovery of the functional gene product. The polypeptide of interest is fused to the carboxyl terminus of glutathione S-protein transferase from Schistosoma japonicum and is easily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion protein can be recovered by elution with glutathione under mild conditions. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of a recognition site for a site-specific protease upstream of this domain. For example, proteins expressed in pGEX-2T plasmids can be cleaved by thrombin; those expressed in pGEX-3X can be cleaved by factor Xa.

[0099] Pharmaceutical Compositions and Administration The present disclosure features pharmaceutical compositions comprising the VLPs described herein. Pharmaceutical compositions useful herein contain a pharma- ceutical acceptable carrier, including any pharmaceutical agent that does not itself induce the production of an immune response harmful to a vertebrate receiving the composition, and a carrier that can be administered without undue toxicity, including any suitable diluent or excipient, and the VLPs of the present disclosure. As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency for use in mammals, more particularly in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias. These compositions can be useful as vaccine and / or antigen compositions for inducing a protective immune response in vertebrates.

[0100] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. A thorough discussion of pharma-ceutically acceptable carriers, diluents, and other excipients is published in Remington's Pharmaceutical Sciences (Mack Pub. Co. NJ, current edition). The formulation should be suitable for the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans and is preferably sterile, non-particulate, and / or non-pyrogenic.

[0101] If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The composition can be in the form of a solid, such as lyophilized powder suitable for reconstitution, or in the form of a liquid, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.Oral preparations can contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0102] In certain embodiments, the VLP compositions are supplied in liquid form, eg, in a hermetically sealed container indicating the quantity and concentration of the VLP composition.

[0103] Preferably, the VLP composition in liquid form is supplied at at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml of the sealed container.

[0104] Alternatively, the vaccine formulation is administered intranasally, either by instillation, large particle aerosol (greater than about 10 microns) or spray to the upper respiratory tract, or small particle aerosol (less than 10 microns) or spray to the lower respiratory tract.While any of the above delivery routes will result in an immune response, intranasal administration offers the added advantage of eliciting mucosal immunity at the site of entry for many viruses, including alphaviruses, e.g., CHIKV or VEEV.

[0105] Thus, the present disclosure also includes a method of formulating a vaccine or antigenic composition that induces immunity against an infection in a mammal or at least one symptom thereof, comprising adding an effective dose of a VLP, such as an alphavirus (e.g., CHIKV or VEEV), to the formulation.

[0106] In certain cases, a single dose of immune stimulation is preferred, but additional dosages can be administered by the same or different route to achieve the desired effect. In neonates and young children, for example, multiple doses may be required to induce a sufficient level of immunity. Administration may continue at intervals throughout childhood as necessary to maintain a sufficient level or protection.

[0107] Similarly, adults who are particularly susceptible to repeated or severe infections, such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary or immune systems, may require multiple immunizations to establish and / or maintain a protective immune response. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing secretory and serum antibodies, and dosages can be adjusted or vaccinations repeated, as necessary, to induce and maintain the desired level of protection.

[0108] The dosage of the pharmaceutical preparation can be easily determined by those skilled in the art, for example, by first identifying the effective dose to induce a preventive or therapeutic immune response, for example, by measuring the serum titer of virus-specific immunoglobulin, or by measuring the inhibition rate of antibodies in serum samples or urine samples or mucosal secretions. The dosage can be determined from animal studies. A non-limiting list of animals used to study the efficacy of vaccines includes guinea pigs, hamsters, ferrets, chinchillas, mice and cotton rats, as well as non-human primates. Most animals are not natural hosts for infectious agents, but can still be useful in studying various aspects of disease. For example, any of the above animals can be dosed with a vaccine candidate, for example, the VLPs of the present disclosure, to partially characterize the induced immune response and / or determine whether any neutralizing antibodies are produced. For example, many studies have been performed in mouse models, since mice are small and their low cost allows researchers to carry out studies on a large scale.

[0109] In addition, human clinical studies can be carried out by those skilled in the art to determine the preferred effective dose for humans. Such clinical studies are routine and well known in the art. The exact dose to be used will also depend on the route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal test systems.

[0110] Also, as is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of immune response, known as adjuvants. Adjuvants have been used empirically to promote a general boost of immunity to unknown antigens. Adjuvants have been used in immunization protocols to boost responses for many years, and are therefore well known to those skilled in the art. Some adjuvants affect the manner in which antigens are presented. For example, immune responses are boosted when protein antigens are precipitated by alum. Emulsification of antigens also extends the period of antigen presentation. The inclusion of any adjuvants described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," which is incorporated herein by reference in its entirety for all purposes, is contemplated within the scope of the present invention.

[0111] Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response that contains killed Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene / Tween-80 emulsion, is also contemplated. MF-59, Novasomes®, MHC antigens can also be used.

[0112] The VLPs of the present disclosure can also be formulated with "immunostimulants." These are the body's own chemical messengers (cytokines) to enhance the immune system's response. Immunostimulants include, but are not limited to, various cytokines, lymphokines and chemokines with immunostimulatory, immune enhancing and proinflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors [e.g., granulocyte macrophage (GM) colony stimulating factor (CSF)]; and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. Immunostimulatory molecules may be administered in the same formulation as the VLPs or separately. Either the protein or an expression vector encoding the protein can be administered to generate an immunostimulatory effect. Thus, in one embodiment, the present disclosure includes antigen and vaccine formulations that include adjuvants and / or immunostimulants.

[0113] According to the present disclosure, the composition comprises: Treating or preventing inflammatory diseases, including cancer or liver fibrosis; raising antibodies against galectins in mammals; Modulating immune responses in controls; Immunostimulation in controls; Inhibiting the interaction between galectins and galectin receptors; or Inhibits galectin activity, It is useful for immunotherapy.

[0114] Examples of cancers that can be treated by the present compositions include, but are not limited to, head and neck cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, cutaneous or intraocular malignant melanoma, melanoma, breast cancer, uterine cancer, ovarian cancer, rectal cancer, colon cancer, duodenal cancer, anal cancer, stomach cancer, liver cancer, testicular cancer, fallopian tube cancer, uterine and cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue cancer, and the like. These include sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, prostate cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, and combinations thereof.

[0115] In particular, when at least one galectin antigen is a galectin-1 epitope peptide, such as Gal-1 epitope peptide #5, #7, #12 or #14 below, especially Gal-1 epitope peptide #5 or #7, the VLPs provided by the present disclosure can be used to treat or prevent cancer, such as lung cancer, and diseases or conditions caused by HPV infection.

[0116] When at least one galectin antigen is a galectin-3 epitope peptide, such as Gal-3 epitope peptide #17, #21, #22 or #23 below, especially Gal-3 epitope peptide #17, #21 or #22, the VLPs provided by the present disclosure can be used to treat or prevent diseases or conditions associated with fibrosis, including, but not limited to, pulmonary fibrosis, liver fibrosis, including non-alcoholic steatohepatitis (NASH), systemic sclerosis and myocardial fibrosis.

[0117] The present application will now be described in detail with reference to the following examples, which are not intended to limit the scope of the present application. [Example 1]

[0118] Preparation of Gal-1 VLPs material and method Galectin-1 (Gal-1) vaccine epitopes were designed by using the amino acid sequences and 3D structures of human and mouse Gal-1 proteins (Table 1). In Table 1, epitope peptides #5-#10 are fragments of human gal-1, and epitope peptides #12 and #14 are fragments of mouse gal-1 protein. To present Gal-1 epitope peptides on the surface of protein particles, the E3 region of the viral structural protein of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) was modified with Gal-1 epitope peptides. The epitope peptides were fused between H at position 321 and S at position 326 of SEQ ID NO:2 for CHIKV, and between G at position 330 and S at position 335 of SEQ ID NO:3 for VEEV, via linkers SGG (N-terminus) and GGS (C-terminus).

[0119] For the generation of plasmid DNA vectors of mammalian expression vectors, pCHIKV-Gal-1 or pVEEV Gal-1, synthetic dsDNA fragments of Gal-1 epitope peptide sequences (gBlocks, IDT) were inserted into specific positions of CHIKV E3 envelope protein DNA in pCHIKV or pVEEV vectors. pCHIKV or pVEEV vectors without any insertion of Gal-1 epitope peptide were used as control VLP expression vectors.

[0120] [Table 1]

[0121] FreeStyle 293F cells (Thermo Fisher Scientific, #R790-07) were cultured in suspension with FreeStyle 293 Expression medium (Thermo Fisher Scientific, #12338-018) at 37°C in the presence of 8% CO2 in a shaking incubator (INFORS HT). 293F cells were transfected with pCHIKV Gal-1 #7, #12 or control plasmid DNA expression vectors at 0.8 μg / 10 DNA with PEI (polyethyleneimine, Polysciences, #23966). 6 cells, DNA:PEI=1:3, and 1.25 × 10 6 Gal-1 VLPs were transfected at 1000 μg / mL of culture medium. Culture supernatants were harvested 4 days after transfection and clarified by centrifugation (3,000 rpm, 10 min) followed by filtration through a 0.45 μm PES membrane filter system (VWR #10040-470). Gal-1 VLP production was confirmed by Western blot analysis of the filtered culture supernatants without any purification or concentration by using rabbit anti-CHIKV antiserum (VLP Therapeutics) or mouse anti-VEEV serum (ATCC) and species-specific HRP-labeled secondary antibodies.

[0122] For purification and enrichment of Gal-1 VLPs, the VLP-containing culture supernatant was layered onto 1.5 mL of OptiPrep (60% w / v) (Iodixanol, Accurate Chemical, #AN1114542) and centrifuged at 52,000 × g for 1.5 h in an SW-28 rotor (Beckman). After removing the supernatant to leave 1.5 mL above the interface, the two layers were mixed and centrifuged at 360,000 × g for 2.5 h in an NVT100 rotor (Beckman) to form a density gradient.

[0123] Crude VLPs were collected and purified by size-exclusion chromatography using a BiologicDuo-Flow FPLC system (Bio-Rad) equipped with a HiPrep 16 / 60 Sephacryl S-500 HR column (GE, #28-9356-06) and phosphate-buffered saline (PBS). The fractions containing VLPs were concentrated with Amicon Ultra-15 centrifugal filter units (EMD Millipore, #UFC910024) and filtered through a 0.20 μm PES membrane.

[0124] Total protein concentration was measured by BCA Protein Assay (Pierce, #23225) according to the manufacturer's instructions. Purity of VLP samples was confirmed by SDS-PAGE analysis (Any kD Mini-PROTEAN TGX Precast Protein Gel, Bio-Rad, #456-9035) followed by Coomassie dye-based staining using QC Colloidal Coomassie Stain (Bio-Rad, #1610803). Sucrose and EDTA were added to the VLP solution to a final concentration of 250 mM and 5 mM, respectively. Protein concentration of Gal-1 VLP samples was adjusted to 0.4 mg / mL and stored at -80°C.

[0125] 293F cells (10 ml) were transfected with 10 ug of plasmids encoding the indicated Gal-1 epitopes into VEEV VLPs. Supernatants were collected and Western blotting was performed to detect anti-VEEV mouse serum (1:1000 dilution). Expression of VLPs carrying Gal-1 epitope peptides #5, #7, #12, and #14 was confirmed in the supernatants (Figure 1). [Example 2]

[0126] Mouse immunogenicity studies material and method For preparation of immunization samples, the VLP solution was thawed and mixed 1:1 (v / v) with 2% Alhydrogel adjuvant (10mg / ml aluminum, Brenntag, #250-843261, batch #5014). Mice (8-week-old female, BALB / c, Envigo) were injected with 10ug (protein) in 0.05mL intramuscularly in the thigh on days 1, 4, and 8. To evaluate the effect of peptide-VLP fusion proteins on antibody titers against self-antigens, 10ug of full-length recombinant human or mouse Gal-1 protein (Sino Bioscience) with Alhydrogel adjuvant was injected into two different groups of mice as controls. Blood samples were collected via submandibular vein on days 0 and 15, and by cardiac puncture on day 29. Animal handling, immunizations, blood collection, and serum preparation were performed at Smithers Avanza Toxicology Service (Gaithersburg, MD) in accordance with IACUC guidelines. Whole blood samples were collected into Serum Separation Tubes (Greiner, #450472) and centrifuged at 1,200 × g for 5 min to separate serum. Collected serum was stored at -20°C until analysis.

[0127] ELISA assay Gal-1 specific serum antibody titers were determined by indirect ELISA. Immunoassay plates (Thermo Fisher Scientific, Maxisorp, #442404) were coated with 100 μL (500 ng / mL) of recombinant human or mouse galectin-1 protein (Sino Bioscience, #10290-HNAE and #50100-MNAE) in PBS overnight at 4°C. After rinsing with TBS-T, wells were blocked with 350 μL of blocking buffer (5% skim milk in TBS-T) for 1 h at room temperature. Wells were washed three times with TBS-T. Mouse sera were diluted 1:50 and further serially diluted 1:5 in blocking buffer to a final dilution of 1:781,250. Diluted sera were incubated in the coated wells at room temperature for 1 h. Wells were washed five times with 400 μL TBS-T and further incubated with HRP-conjugated anti-mouse IgG+M+A antibody (Thermo Fisher, #PA1-84388) for 30 min at room temperature. Plate wells were washed five times with TBS-T and incubated with 100 μL HRP substrate (SureBlue TMB 1-Component Microwell Peroxidase Substrate, Sera Care, #5120-0076). The reaction was stopped with 100 μL stop solution (1M H2SO4) and absorbance was measured at 450 nm by a microplate reader (Synergy / HTX, BioTek). Data were analyzed using Microsoft Excel (Microsoft) and GraphPad Prism 8.0 (GraphPad Software). The endpoint titer was defined as the higher serum dilution that produced an absorbance reading higher than the blank value plus three standard deviations.

[0128] The immunoglobulin class and subclass of anti-Gal-1 antibodies in immune sera were determined by indirect ELISA and subclass-specific secondary antibody panel. Immunoassay plates were coated with 100 μL (500 ng / mL) of recombinant mouse galectin-1 protein (Sino Bioscience, #50100-MNAE) in PBS overnight at 4°C. After rinsing with TBS-T, wells were blocked with 350 μL of blocking buffer (5% skim milk in TBS-T) for 1 h at room temperature. Wells were washed three times with TBS-T. Mouse sera were diluted 1:1000 in blocking buffer. Each diluted serum was transferred to eight different Gal-1-coated wells and incubated at room temperature for 1 h. The wells were washed five times with 400 μL of TBS-T and further incubated with Mouse Typer Sub Isotyping Panel of HRP-conjugated anti-mouse Ig subclass specific antibodies (Bio-Rad, #172-2055) for 30 min at room temperature. The plate wells were washed five times with TBS-T and incubated with 100 μL of HRP substrate (SureBlue™ TMB 1-Component Microwell Peroxidase Substrate, Sera Care, #5120-0076). The reaction was stopped with 100 μL of stop solution and the absorbance at 450 nm was measured by a microplate reader.

[0129] result Through screening of Gal-1 peptide fragments (listed in Table 1), we identified two promising Gal-1 vaccine candidates, #5 and #7, which induced anti-human Gal-1 antibody combination with the CHIKV virus-like particle platform (Figure 2A). The immunogenicity of Gal-1 VLPs in naive BALB / c mice was measured by ELISA for mouse Gal-1 and human Gal-1, respectively. Four weeks after the first injection, anti-human and anti-mouse galectin-1 antibodies were induced in mice immunized with Gal-1 VLPs. For proof-of-concept studies in mouse models, the peptide sequence of Gal-1 VLP #5, derived from human Gal-1, was modified with mouse Gal-1 sequence to obtain Gal-1 VLP #12. Binding of rhGal-1 by sera immunized with Gal-1 #5 VLP was preferential over rmGal-1 binding (Figure 2B). On the other hand, binding of rmGal-1 by sera immunized with Gal-1 #12 VLP was preferential over rhGal-1 binding.

[0130] For efficient production of Gal-1 VLPs, the origin of VLPs was changed from chikungunya virus (CHIKV) to Venezuelan equine encephalitis virus (VEEV). A total of five different particles were prepared, including four Gal-1 VEEV VLPs (Table 1, #5, #7, #12, and #14) and a control VEEV VLP. The molecular size and purity of Gal-1 VLPs and control VLPs were analyzed by SDS-PAGE under denaturing conditions (Figure 3A). All VLP samples were consistent with the theoretical molecular weight and had a purity of more than 90%. The protein bands between 50 and 75 kDa are fusion proteins of Gal-1 peptide and viral structural proteins. The molecular weight varies depending on the length of the inserted Gal-1 peptide. Mice were intramuscularly injected with 10 μg of Gal-1 VLP protein with aluminum hydroxide adjuvant three times a week. Four weeks after immunization, anti-Gal-1 antibody titers specific for human or mouse Gal-1 were determined by ELISA, respectively. The four Gal-1 peptide conjugate VLP molecules #5, 7, 12, and 14 induced anti-mouse Gal-1 antibodies in mice, but the recombinant mouse Gal-1 protein did not (Fig. 3D, E). Gal-1 #5 and #7 are different human Gal-1 epitope sequence peptides, and VLPs carrying these peptides induced anti-human Gal-1 antibodies. Gal-1 VLP #12 and #14 are different mouse Gal-1 epitope peptides, and VLPs carrying these peptides efficiently induced anti-mouse Gal-1 antibodies (Fig. 3E). Gal-1 peptides #5 and #12 encode four β-sheets S3, S4, S5, and S6 near the carbohydrate recognition domain (CRD) (Fig. 3B). Gal-1 peptides #7 and #14 encode β-sheets F4 and F5 that may be important in homophilic dimerization (Fig. 3C). Chimeric antigens of self-proteins with virus-derived proteins displayed on VLP protein nanoparticles break immune tolerance to self-proteins and efficiently stimulate antibody production. Although Gal-1 proteins are highly conserved among species (Figure 3F), sera immunized with Gal-1 epitope peptides from different species induced selective anti-Gal-1 antibodies in humans or mice (Figure 3E).The Gal-1 VLP vaccine was able to effectively stimulate humoral immune responses against self-proteins. [Example 3]

[0131] In vivo tumor models material and method Animal handling, immunizations, blood sampling, tumor inoculation, tumor measurements, and serum preparation were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee under the supervision of Dr. Judith Varner, Professor of Pathology and Medicine, Moores Cancer Center University of California, San Diego (La Jolla, CA). Tumor size was measured twice weekly. Tumor volume was calculated as D×d2 / 2, where D=longest diameter and d=shortest diameter. Mice were sacrificed on day 41 and blood and tumor tissues were collected. Endpoint tumor weights were measured. All data collected in the study were analyzed by Microsoft Excel and GraphPad Prism.

[0132] LLC Model C57BL6 (female, 8 weeks old) mice were immunized intramuscularly with 10 μg of CHIKV Gal-1 virus-like particle vaccine with 2% Alhydrogel adjuvant at 1:1 (v / v) three times on days 1, 4, and 8 (n=8). Control mice were injected with PBS containing 2% Alhydrogel. On day 15, 200 μl of blood was collected from each animal prior to tumor inoculation. Blood was processed as plasma and stored at -20°C. Mice were challenged subcutaneously (sc) in the flank with Lewis lung carcinoma cells (1×106 cells / 100 μL) in serum-free PBS.

[0133] MEER HPV Model C57BL6 (female, 8 weeks old) mice were immunized intramuscularly with 10 μg of VEEV Gal-1 virus-like particle vaccine with 2% Alhydrogel adjuvant 1:1 (v / v) three times on days 1, 4, and 8, and weekly from day 15 onwards. Control mice were injected with control VEEV VLP vaccine with 2% Alhydrogel. On day 15, 200 μl of blood was collected from each animal prior to tumor inoculation. Blood was processed as plasma and stored at -20°C. Mice were inoculated with MEER HPV+ cells (1×10 6 The mice were exposed to 100 μL (cells / 100 μL) subcutaneously (sc) in the flank.

[0134] result The effect of Gal-1 VLP vaccine on cancer therapy was examined in a mouse model of lung cancer. C57BL6 mice were pre-immunized with control VLP or Gal-1 VLP 2 weeks before tumor inoculation. Anti-mouse Gal-1 antibody titers were determined by ELISA (Figure 4A). Antibody titers against viral proteins were also examined as a reference for immunity (Figure 4B). Mice receiving Gal-1 VLP produced anti-mouse Gal-1 antibodies, as previously observed in different mouse strains. Mice immunized with the two different Gal-1 VLPs preferentially expressed IgG2a anti-mouse Gal-1 antibodies (Figure 4C).

[0135] Lewis lung carcinoma cells were inoculated subcutaneously on day 15, and tumor growth was monitored. There was no statistically significant difference in tumor volume (Figure 5A-D) and endpoint tumor weight (Figure 5E) between the control and VLP-treated groups, but tumor growth in mice treated with Gal-1 #12 was slower than that in the other groups.

[0136] We investigated the effect of Gal-1 vaccine on a mouse HPV+ head and neck squamous cell (Square) cancer cell model. Tumor growth in mice treated with Gal-1 #14 VLP vaccine was slower than that in controls, but there was no statistically significant difference between either group (Figures 6A and 6B). [Example 4]

[0137] Preparation of VLPs carrying Gal-3 antigen material and method Galectin-3 (Gal-3) epitope peptides were designed by using the protein amino acid sequences and 3D structures of human and mouse Gal-3 proteins (Table 2). For the generation of mammalian expression vector, pVEEV Gal-3 plasmid DNA vector, a synthetic dsDNA fragment of Gal-3 epitope peptide sequence (gBlocks, IDT) was inserted into a specific position of DNA encoding VEEV E3 envelope protein in pVEEV vector (between G at position 330 and S at position 335). pVEEV vector without any insertion of Gal-1 epitope peptide was used as a control VLP expression vector.

[0138] FreeStyle 293F cells (Thermo Fisher Scientific, #R790-07) were cultured in suspension with FreeStyle 293 Expression medium (Thermo Fisher Scientific, #12338-018) at 37°C with 8% CO2 in a shaking incubator. 293F cells were transfected with pVEEV Gal-3 VLP or control plasmid DNA expression vectors at 0.8 μg / 10 DNA with PEI (polyethyleneimine, Polysciences, #23966). 6 cells, DNA:PEI=1:3 (w / w), and 1.25 × 10 6 The cells were transfected at 1 mL of culture medium. Culture supernatants were harvested 4 days after transfection and clarified by centrifugation (3,000 rpm, 10 min) followed by filtration through a 0.45 μm PES membrane filter system (VWR #10040-470).

[0139] The VLP-containing culture supernatant was layered on top of 1.5 mL of OptiPrep (60% w / v) (Iodixanol, Accurate Chemical, #AN1114542) and centrifuged at 52,000 × g for 1.5 h in an SW-28 rotor (Beckmann). After removing the supernatant to leave 1.5 mL above the interface, the two layers were mixed to make a 50% OptiPrep solution and centrifuged at 360,000 × g for 2.5 h in an NVT100 rotor (Beckman) to form a density gradient.

[0140] Crude VLPs were collected and purified by size-exclusion chromatography using a Biologic Duo-Flow FPLC system (Bio-Rad) equipped with a HiPrep 16 / 60 Sephacryl S-500 HR column (GE, #28-9356-06) and phosphate-buffered saline (PBS). The VLP-containing fraction was concentrated with an Amicon Ultra-15 centrifugal filter unit (EMD Millipore, #UFC910024) and filtered through a 0.20 μm PES membrane.

[0141] Total protein concentration was measured by BCA Protein Assay (Pierce, #23225) according to the manufacturer's instructions. Purity of VLP samples was confirmed by SDS-PAGE analysis (Any kD Mini-PROTEAN TGX Precast Protein Gel, Bio-Rad, #456-9035) followed by Coomassie dye-based staining using QC Colloidal Coomassie Stain (Bio-Rad, #1610803). Sucrose and EDTA were added to the VLP solution to a final concentration of 250 mM and 5 mM, respectively. Protein concentration of VLP samples was adjusted to 0.4 mg / mL and stored at -80°C.

[0142] [Table 2] The molecular size and purity of VEEV VLPs containing Gal-3 epitope peptides and control VEEV VLPs were analyzed by SDS-PAGE under denaturing conditions (Figure 7). All VLP samples were consistent with the theoretical molecular weight and were more than 90% pure. Protein bands between 50 and 75 kDa are fusion proteins of Gal-3 peptides with VEEV viral structural proteins, and the size variations depend on the length of the peptide. [Example 5]

[0143] Mouse immunogenicity studies material and method For preparation of immunization samples, the VLP solution was thawed and mixed 1:1 (v / v) with Alhydrogel adjuvant (10 mg / ml aluminum, Brenntag, #250-843261, batch #5014). Mice (8-week-old female, BALB / c, Envigo) were injected with 10 ug (protein) in 0.05 mL intramuscularly in the thigh on days 1, 4, and 8. Blood samples were collected via the submandibular vein on days 0 and 15 and by cardiac puncture on day 29. Animal handling, immunizations, blood collection, and serum preparation were performed by Smithers Avanza Toxicology Service (Gaithersburg, MD) in accordance with IACUC guidelines. Whole blood samples were collected into Serum Separation Tubes (Greiner, #450472) and centrifuged at 1,200×g for 5 minutes to separate serum. The collected serum was stored at -20°C until analysis.

[0144] ELISA assay Gal-3 specific serum antibody titers were determined by indirect ELISA. Immunoassay plates (Thermo Fisher Scientific, Maxisorp, #442404) were coated with 100uL (500ng / mL) of recombinant human or mouse galectin-3 protein (GA3-H5129, AcroBioscience and #599806, Biolegend) in PBS overnight at 4°C. After rinsing with TBS-T, wells were blocked with 350uL of blocking buffer (5% skim milk in TBS-T) for 1 hour at room temperature. Wells were washed three times with TBS-T. Mouse sera were diluted 1:50 and further serially diluted 1:5 in blocking buffer to a final dilution of 1:781,250. Diluted sera were incubated in the coated wells at room temperature for 1 hour. The wells were washed five times with 400uL TBS-T and further incubated with HRP-labeled anti-mouse IgG+M+A antibody (Thermo Fisher, #PA1-84388) for 30 minutes at room temperature. The plate wells were washed five times with TBS-T and incubated with 100uL HRP substrate (SureBlue™ TMB 1-Component Microwell Peroxidase Substrate, Sera Care, #5120-0076). The reaction was stopped with 100uL stop solution and absorbance was measured at 450nm by a microplate reader (Synergy / HTX, BioTek). Data were analyzed using Microsoft Excel (Microsoft) and GraphPad Prism 8.0 (GraphPad Software). The endpoint titer of mouse serum was determined by the highest dilution whose OD was higher than 3xSD of assay control plus control.

[0145] result The immunogenicity of Gal-3 VLP in naive BALB / c mice was determined by ELISA. Four weeks after the first injection, anti-human and anti-mouse galectin-3 antibodies in the serum of mice immunized with Gal-3 VLP were analyzed using recombinant mouse or human Gal-3 proteins. The results are shown in Figure 8. Gal-3 VLP #17 consists of a human N-terminal Gal-3 epitope peptide and induced more anti-human Gal-3 antibodies than anti-mouse Gal-3. In contrast, Gal-3 VLP #23 encodes a mouse Gal-3 N-terminal peptide and induced more anti-mouse Gal-3 antibodies than anti-human Gal-3 antibodies. Gal-3 VLP #21 and 22 consist of a human Gal-3 N-terminal repeat epitope peptide, with #22 having a longer repeat sequence than that of #21. [Example 6]

[0146] Flow cytometric analysis of serum antibodies binding to cell surface Gal-3 material and method 293F cells (2 × 10 in FreeStyle 293 culture medium) 6Cells / mL) were incubated with recombinant human galectin-3 (30 μg / mL) for 30 min at room temperature. Cells were spun down and washed twice with cell culture medium. Pelleted cells were resuspended in FC staining buffer (5% FBS in PBS) and 100 μL was transferred to a 96-well V-bottom plate. Plates were centrifuged at 800×g to pellet cells and the supernatant was discarded. Serum from 5 mice was pooled and diluted 1:250 with FC staining buffer. Diluted serum was added to wells with pelleted cells with and without Gal-3 treatment. Cells were resuspended by pipetting and incubated for 1 h at room temperature. After incubation, cells were washed by pelleting and resuspending in FC staining buffer. The washing step was repeated twice and cells were resuspended in Alexa 488-labeled goat anti-mouse IgG+M antibody (Invitrogen, 1:500 dilution) and incubated for 30 min at room temperature protected from light. The cells were washed twice with FC staining buffer, resuspended in 200 μL of FC staining buffer, and analyzed by flow cytometer (Attune Acoustic Focusing Flow Cytometer, Thermo Fisher).

[0147] result The recognition and binding of anti-Gal-3 antibodies to cell surface Gal-3 protein was examined by flow cytometry analysis. Endogenous expression of Gal-3 protein in 293F cells was not detectable by Western blotting analysis. Single cell suspensions of 293F cells were incubated with recombinant human Gal-3 protein and then washed three times to remove unbound protein. Gal-3 VLP-immunized serum was diluted in blocking buffer, followed by fluorescently labeled anti-mouse IgG+M. Mouse serum immunized with control VLP was used as a control. Both serum from mice immunized with Gal-3 #17 and #22 bound to Gal-3-treated cells. (Figure 9) [Example 7]

[0148] Pull-down assay material and method 20μL of immune mouse serum, 20μg of mouse anti-Gal-3 monoclonal antibody (cl.B2C10, Santa Cruz), or 20μg of mouse control IgG (Biolegend) were diluted to 1mL in PBS and incubated with 40μL of Protein G magnetic beads (GenScript) for 1h at room temperature on a rotator. The beads are washed 3 times with PBS on a magnetic rack. The coated magnetic beads are resuspended in 1mL of recombinant human Gal-3 solution (2ug / mL) in the presence or absence of 50mM lactose and incubated for 1h at room temperature on a rotator. The beads are then washed 3 times with PBS and boiled with 50μL of 1x Laemmli SDS-PAGE sample buffer containing 2-mercaptoethanol. Samples were separated by SDS-PAGE and transferred to PVDF membranes. Gal-3 protein was probed with anti-Gal-3 monoclonal antibody (cl. Gal397, BioLegend) and HRP-conjugated anti-mouse IgG. The membrane was incubated with Clarity Western ECL Substrate (Bio-Rad) and images were captured by a ChemiDoc XRS+ imaging system (Bio-Rad).

[0149] result The neutralizing ability of anti-Gal-3 antibodies induced by Gal-3 VLP vaccine was examined by pull-down assay. Recombinant Gal-3 protein floating in solution was captured by anti-Gal-3 antibodies linked to protein G magnetic beads. Gal-3 protein was pulled down by monoclonal Gal-3 antibody B2C10 and antisera from Gal-3 VLP-vaccinated mice, but not by control VLPs. Gal-3 VLPs effectively induced high titer anti-Gal-3 antibodies that recognized the membrane-bound and floating forms of Gal-3 protein. (Figure 10) [Example 8]

[0150] Mouse NASH model material and method C57BL / 6 mice (female, 14 days pregnant) were obtained from Japan SLC Co., Ltd. (Japan). All animals used in the study were housed and maintained in accordance with the Japan Pharmaceutical Association guidelines for animal use. Animals were kept in an SPF facility under controlled conditions of temperature (23 ± 2°C), humidity (45 ± 10%), lighting (12-h artificial light-dark cycle; light on from 8:00 to 20:00) and ventilation.

[0151] NASH was induced in 25 male mice by a single subcutaneous injection of a solution of 200 μg of streptozotocin (STZ, Sigma-Aldrich, USA) at 2 days of age and feeding with a high fat diet (HFD, 57 kcal% fat, Cat#HFD32, Nippon Clea Co., Ltd., Japan) after 4 weeks of age. Gal-3 VLP, control VLP and vehicle were administered intramuscularly (thigh) in a volume of 50 μL / body. The leg of injection was changed each time to avoid local inflammation. Survival, clinical signs and behavior were monitored daily. Body weight was recorded before treatment. Mice were observed for significant clinical signs of toxicity, moribundity and death approximately 60 minutes after each administration. Animals were sacrificed at 9 weeks of age by exsanguination via direct cardiac puncture under isoflurane anesthesia (Pfizer Inc.).

[0152] For serum samples, nonfasting blood was collected from the facial vein into anticoagulant-free serum separator tubes on day 15 (6 weeks of age). The collected blood was centrifuged at 3,500 × g for 5 min at 4 °C. 20 μL of the supernatant was collected and stored at -80 °C for biochemistry. Serum ALT levels were measured by FUJI DRI-CHEM 7000 (Fujifilm Corporation, Japan). For HE staining, sections were cut from paraffin blocks of liver tissue prefixed with Bouin's solution and stained with Lillie-Mayer's Hematoxylin (Muto Chemical Co., Japan) and eosin solution (Fujifilm Wako Pure Chemical Corporation). Statistical analysis was performed using the Bonferroni Multiple Comparison Test on GraphPad Prism 6 (GraphPad Software Inc., USA). A P value of <0.05 was considered statistically significant. Trends or tendencies were assumed if a one-tailed t-test returned a P value < 0.1. Results were expressed as mean ± SD.

[0153] result The effect of Gal-3 inhibition by Gal-3 VLP vaccine was examined in a murine STAM nonalcoholic steatohepatitis (NASH) model. Mice treated with STZ and fed a high-fat diet develop nonalcoholic fatty liver disease (NAFLD) and NASH at 6-8 weeks of age. Mice treated with control or Gal-3 VLP were given 3 injections in the first week, then weekly until 8 weeks of age, for a total of 6 injections, starting at 4 weeks of age.

[0154] There were no significant differences in body weight change, end-point liver weight, and kidney weight observed between any two groups. One mouse in group 5 was found dead on study day 23, but this was likely an accidental death and was not related to toxicity of the treatment. At the end-point of the study, serum alanine aminotransferase (ALT) levels were measured as a general marker of liver damage. ALT levels in the study rats were higher than that of normal levels (17.5-30.2 U / L) (Johnson-Delaney 1996). There were no significant differences in ALT between any of the groups. To evaluate the effect of Gal-3 vaccine on NASH disease progression, the NAFLD activity score (NAS) was calculated according to the Kleiner criteria (Kleiner, et al. 2005). There are three different component assessments of NAS in steatosis, lobular inflammation, and hepatocyte ballooning (Table 3, Figure 11A-C). H&E stained liver sections from the study in NASH model were scored and analyzed (Table 4). Liver sections from the vehicle group showed microvesicular and macrovesicular fatty deposits, hepatocyte ballooning, and inflammatory cell infiltration. NAS in the control VLP group and Gal-3 #23 VLP group tended to be lower compared to the vehicle group (Figure 11, Table 4). Ballooning scores, especially in hepatocytes, were reduced by VLP vaccine. There was no statistically significant difference in total NAS scores between any of the groups involved, but a trend toward lower ballooning scores was observed in the groups treated with control VLP or Gal-3 vaccine.

[0155] [Table 3]

[0156] [Table 4]

Claims

1. A virus-like particle comprising an alphavirus viral structural protein and at least one galectin antigen.

2. The virus-like particle of claim 1 , wherein the viral structural proteins are derived from an alphavirus.

3. 3. The virus-like particle of claim 2, wherein the viral structural proteins are derived from Chikungunya virus or Venezuelan equine encephalitis virus.

4. The virus-like particle of claim 3 , wherein the viral structural proteins include capsid, E1, E2, and E3.

5. 4. The virus-like particle of claim 3, wherein the viral structural proteins are derived from Chikungunya virus strain 37997 or strain OPY-1, or Venezuelan Equine Encephalitis virus strain TC-83.

6. The virus-like particle of claim 1 , wherein at least one galectin antigen is inserted into the envelope protein E3.

7. The virus-like particle of claim 5, wherein the at least one galectin antigen is inserted between residues 321 and 326 of SEQ ID NO:1 or SEQ ID NO:2, or between residues corresponding to residues 321 and 326 of SEQ ID NO:1 or SEQ ID NO:2, or between residues 330 and 335 of SEQ ID NO:3, or between residues corresponding to residues 330 and 335 of SEQ ID NO:

3.

8. The virus-like particle of claim 1, wherein the at least one galectin antigen is a peptide fragment of galectin-1.

9. The at least one galectin antigen is: #5: SFVLNLGKDSNNLSLHFNPRFNAHGDANTIVSNSKDGGAWGTEQREAVFPFQPGS #7: ANLTVKLPDGYEFKFPNRLNLEA #12: SFVLNLGKDSNNLSLHFNPRFNAHGDANTIVSNTKEDGTWGTEHREPAFPFQPGS, and #14: ADLTIKLPDGHEFKFPNRLNMEA The virus-like particle of claim 8, selected from the group consisting of:

10. The virus-like particle of claim 1, wherein the at least one galectin antigen is a peptide fragment of galectin-3.

11. The at least one galectin antigen is: #17: ADNFSLHDALSGSGNPNPQGWPGAWGNQPA #21: YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGA #22: YPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGA, and #23: ADSFSLNDALAGSGNPNPQGYPGAWGNQPA The virus-like particle of claim 10, selected from the group consisting of:

12. 12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the virus-like particle of any one of claims 1 to 11.

13. A vector comprising the nucleic acid molecule of claim 12, Optionally, an expression control sequence is operably linked to the nucleic acid molecule. vector.

14. (a) the virus-like particle according to any one of claims 1 to 11, an isolated nucleic acid molecule comprising a nucleotide sequence encoding said virus-like particle, and / or a vector comprising said nucleic acid molecule, optionally comprising an expression control sequence operably linked to said nucleic acid molecule; and (b) a pharma- ceutically acceptable carrier; 20. A pharmaceutical or vaccine composition comprising:

15. The composition described in claim 14 for immunotherapy targeting galectin.

16. Treating or preventing cancer or inflammatory diseases; Raising antibodies against galectins in mammals; Modulating immune responses; Immunostimulation; Inhibits interactions between galectins; or The composition according to claim 15 for inhibiting galectin activity.

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