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WO2024248287A1 - Composition pharmaceutique pour le traitement de maladies inflammatoires comprenant un polypeptide dérivé de mycobacterium tuberculosis - Google Patents

Composition pharmaceutique pour le traitement de maladies inflammatoires comprenant un polypeptide dérivé de mycobacterium tuberculosis Download PDF

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WO2024248287A1
WO2024248287A1 PCT/KR2024/003334 KR2024003334W WO2024248287A1 WO 2024248287 A1 WO2024248287 A1 WO 2024248287A1 KR 2024003334 W KR2024003334 W KR 2024003334W WO 2024248287 A1 WO2024248287 A1 WO 2024248287A1
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disease
nlrp3
polypeptide
variant
amino acid
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Korean (ko)
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양철수
김효근
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Industry University Cooperation Foundation IUCF HYU
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Industry University Cooperation Foundation IUCF HYU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)

Definitions

  • the present invention relates to a polypeptide derived from Mycobacterium tuberculosis that specifically binds to NLRP3, and a pharmaceutical composition comprising the same for treating inflammatory diseases.
  • inflammasomes There are four known inflammasomes, namely NLRP1, NLRP3, NLRP4, and Aim2 inflammasomes, all of which contain PRRs belonging to the NLR family.
  • NLRP3 inflammasome plays a pivotal role in shaping immune responses and regulating the integrity of intestinal homeostasis in many common inflammatory diseases.
  • NLRP3 a multiprotein complex consisting of the NLRP3 scaffold, the adaptor apoptosis speck-like protein (ASC), and the effector procaspase-1, initiates the formation of the inflammasome by interacting with ASC, which then recruits and activates procaspase-1 to produce active caspase-1 and converts the cytokine precursors pro-IL-1 ⁇ and pro-IL-18 into mature biologically active IL-1 ⁇ and IL-18, respectively. Once activated, active IL-1 ⁇ and IL-18 induce a series of inflammatory responses and pyogenic cell death.
  • ASC adaptor apoptosis speck-like protein
  • the NLRP3 inflammasome is produced by bone marrow-derived macrophages upon stimulation by microbial and nonmicrobial factors, such as bacterial toxins, particulate matter, and lipopolysaccharide (LPS).
  • microbial and nonmicrobial factors such as bacterial toxins, particulate matter, and lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • K + efflux, Ca2 + signaling, mitochondrial dysfunction, and reactive oxygen species (ROS) production for example, particulate matter activates the NLRP3 inflammasome by inducing endocytosis and damage to the lysosomal membrane, leading to the release of cathepsin B into the cytosol.
  • ROS reactive oxygen species
  • the present invention aims to provide a polypeptide capable of treating inflammatory diseases by activating NLRP3 inflammasome.
  • the present invention provides a polypeptide or a variant thereof comprising a sequence represented by SEQ ID NO: 1, which specifically binds to NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) protein.
  • the polypeptide consists of the sequence represented by SEQ ID NO: 1, and in another embodiment of the present invention, the polypeptide comprises the sequence represented by SEQ ID NO: 2, and in yet another embodiment of the present invention, the variant comprises a conservative substitution at a position excluding the 4th, 6th, and 7th amino acids of SEQ ID NO: 1, and in yet another embodiment of the present invention, the conservative substitution is at least one selected from the group consisting of F1Y, Y2F, A3G, F5Y, A8G, L9V, and S10T, or at least one selected from the group consisting of F1Y, Y2F, A3G, F5Y, A8G, L9I, and S10T.
  • the present invention provides an NLRP3-specific binding molecule comprising the above-mentioned polypeptide or a variant thereof.
  • the present invention provides a pharmaceutical composition for treating an inflammatory disease, comprising the polypeptide or variant, and in one embodiment of the present invention, the inflammatory disease is Alzheimer's disease, atherosclerosis, asthma and allergic airway inflammation, gout, inflammatory bowel disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, myocardial infarction, multiple sclerosis or experimental autoimmune encephalitis, nephropathy, post-influenza hyperinflammation, graft-versus-host disease, stroke, silicosis, type 1 diabetes, obesity-induced inflammation or insulin resistance, rheumatoid arthritis, contact hypersensitivity, or traumatic brain injury.
  • the inflammatory disease is Alzheimer's disease, atherosclerosis, asthma and allergic airway inflammation, gout, inflammatory bowel disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, myocardial infarction, multiple sclerosis or experimental autoimmune encephalitis, nephropathy, post-influen
  • the present invention provides a nucleic acid molecule expressing the above polypeptide or variant.
  • the present invention provides a pharmaceutical composition for treating an inflammatory disease, comprising the nucleic acid molecule described above, and in one embodiment of the present invention, the inflammatory disease is Alzheimer's disease, atherosclerosis, asthma and allergic airway inflammation, gout, inflammatory bowel disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, myocardial infarction, multiple sclerosis or experimental autoimmune encephalitis, nephropathy, post-influenza hyperinflammation, graft-versus-host disease, stroke, silicosis, type 1 diabetes, obesity-induced inflammation or insulin resistance, rheumatoid arthritis, contact hypersensitivity, or traumatic brain injury.
  • the inflammatory disease is Alzheimer's disease, atherosclerosis, asthma and allergic airway inflammation, gout, inflammatory bowel disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, myocardial infarction, multiple sclerosis or experimental autoimmune encephalitis, nephropathy, post-
  • the present invention provides a recombinant vector comprising the nucleic acid molecule described above.
  • the present invention provides a host cell comprising the nucleic acid molecule described above.
  • polypeptide according to the present invention inhibits the activation of the NLRP3 inflammasome by specifically binding to the NLRP3 protein. Therefore, the polypeptide according to the present invention, or a variant thereof, or a nucleic acid molecule expressing the same, can be used as a therapeutic agent for inflammatory diseases.
  • Figure 1A shows the results of confirming the binding of NLRP3 to Rv0747/PE_PGRS10
  • Figure 1B shows the results of confirming the binding site to Rv0747/PE_PGRS10 through a deletion mutant of NLRP3
  • Figure 1C shows the results of confirming the binding activity of NLRP3 according to the position of Rv0747/PE_PGRS10
  • Figure 1D is a peptide sequence predicted as a site showing the binding ability to NLRP3 in Rv0747/PE_PGRS10.
  • FIG. 2A is a result confirming the degree of binding between NLRP3 and ASC according to peptide treatment according to one embodiment of the present invention
  • FIGS. 2B and 2C are results confirming the specific secretion inhibition ability for IL-1 ⁇ , IL-18, and caspase-1 according to peptide treatment according to one embodiment of the present invention
  • FIG. 2D is a result confirming that the peptide according to one embodiment of the present invention has superior binding ability to NLRP3 compared to ASC.
  • Figure 3 shows the results of confirming changes in ubiquitination (Figure 3A) and phosphorylation (Figure 3B) of the NRR domain by peptide treatment according to one embodiment of the present invention.
  • Figure 4 is a result of confirming the amino acid positions essential for binding to NLRP3 among peptides according to one embodiment of the present invention.
  • NLRP3 inflammasome is a multiprotein complex that plays a central role in regulating the innate immune system and inflammatory signaling.
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damage-associated molecular patterns
  • NLRP3 oligomerizes and activates caspase-1, which initiates the processing and release of the proinflammatory cytokines IL-1 ⁇ and IL-18.
  • NLRP3 inflammasomes resulting from activation of the NLRP3 inflammasome are readily observed in patients with gain-of-function mutations in NLRP3.
  • CFS cryopyrin-associated periodic syndromes
  • the antagonistic pleiotropic gene NLRP3 is known to provide an advantage in controlling infections early in life, and given the plethora of damage-related stimuli sensed by NLRP3, it is thought to have evolved to initiate immune responses to tissue damage. Whatever evolutionary advantages NLRP3 may have in detecting and combating infections, it is now responsible for many noncommunicable diseases in the elderly population.
  • NLRP3 is the primary sensor for these types of sterile inflammatory signals, and is therefore a key driver of chronic inflammatory tissue responses in a variety of diseases.
  • NLRP3 has been implicated in a diverse range of diseases, but based on a combination of animal model studies and clinical data, it plays a critical role in inflammatory diseases in four broad categories: metabolic dysfunction, diseases resulting from crystal or aggregate formation, acute tissue damage, or fibrosis secondary to chronic inflammation.
  • NLRP3 activation The inflammatory diseases caused by NLRP3 activation that have been studied to date are as follows: Alzheimer's disease (Daniels, M. J. D. et al. Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer's disease in rodent models. Nat. Commun. 7, 1-10 (2016). This is the first paper to demonstrate that the fenamate class of NSAIDs, already approved by the FDA for other treatments, is able to inhibit NLRP3.;Heneka, M. T. et al. NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice. Nature 493, 674-678 (2014).;Dempsey, C. et al.
  • NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J. Hepatol. 66, 1037-1046 (2017).), Myocardial infarction (Marchetti, C. et al. A novel pharmacologic inhibitor of the NLRP3 inflammasome limits myocardial injury after ischemia-reperfusion in the mouse. J. Cardiovasc. Pharmacol. 63, 316-322 (2014).;van Hout, G. P. J. et al. The selective NLRP3-inflammasome inhibitor MCC950 reduces infarct size and preserves cardiac function in a pig model of myocardial infarction. Eur. Heart J.
  • Interferon- ⁇ therapy against EAE is effective only when development of the disease depends on the NLRP3 inflammasome. Sci. Signal. 5, ra38 (2012).), Nephropathy (Mulay, S. R. et al. Calcium oxalate crystals induce renal inflammation by NLRP3-mediated IL-1 ⁇ secretion. J. Clin. Invest. 123, 236-246 (2012).;Ludwig-Portugall, I. et al. An NLRP3-specific inflammasome inhibitor attenuates crystal-induced kidney fibrosis in mice. Kidney Int. 90, 525-539 (2016).), Hyperinflammation after influenza infection (Coates, B. M. et al.
  • Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischemic brain injury. Commun. 6, 1-11 (2015).; NLRP3 deficiency ameliorates neurovascular damage. J. Cereb. Blood Flow Metab. 34, 660-667 (2014).;Ren, H. et al. Selective NLRP3 (pyrin domain-containing protein 3) inflammasome inhibitor reduces brain injury after intracerebral hemorrhage. Stroke 49, 184-192 (2018).), silicosis (Cassel, S. L. et al. The Nalp3 inflammasome is essential for the development of silicosis. Proc. Natl Acad.
  • Traumatic brain injury (Irrera, N. et al. Lack of the Nlrp3 inflammasome improves mice recovery following traumatic brain injury. Front. Pharmacol. 8, 459 (2017).;Ismael, S., Nasoohi, S. & Ishrat, T. MCC950, the selective NLRP3 inflammasome inhibitor protects mice against traumatic brain injury. J. Neurotrauma 35, 1294-1303 (2016).
  • the present invention provides a peptide derived from Mycobacterium tuberculosis, which specifically binds to NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) and can be used as a therapeutic agent for inflammatory diseases by inhibiting the activation of NLRP3 inflammasome. More specifically, the peptide according to the present invention is derived from Rv0747/PE_PGRS10 (SEQ ID NO: 2) of Mycobacterium tuberculosis. Wild-type Rv0747/PE_PGRS10 consists of 801 amino acids.
  • the present invention provides a peptide having a FYAQFVQALS sequence (SEQ ID NO: 1), which consists of the 72nd to 81st amino acids of Rv0747/PE_PGRS10, which specifically binds to NLRP3.
  • the DNA sequence encoding the full-length Rv0747/PE_PGRS10 is represented by SEQ ID NO: 4, and the DNA sequence encoding the full-length Rv0747/PE_PGRS10 is represented by SEQ ID NO: 3.
  • fragment means a part of the amino acid sequence, i.e. a sequence which represents an amino acid sequence that is shortened at the N-terminus and/or the C-terminus.
  • a fragment shortened at the C-terminus can be obtained, for example, by translating a truncated open reading frame lacking the 3'-end of the open reading frame.
  • a fragment shortened at the N-terminus can be obtained, for example, by translating a truncated open reading frame lacking the 5'-end of the open reading frame, as long as the truncated open reading frame comprises an initiation codon which serves to initiate translation.
  • a fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence.
  • the fragment of the amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.
  • a “variant”, “variant”, “variant protein”, or “variant polypeptide” of a polypeptide or protein refers to any analog, fragment, derivative, or mutant derived from the polypeptide or protein and which retains at least one biological property of the polypeptide or protein.
  • Different variants of the polypeptide or protein may exist in nature. These variants may be allelic mutations characterized by differences in the nucleotide sequence of the structural gene encoding the protein, or may include differential splicing or post-translational modifications.
  • One skilled in the art can generate variants having one or more amino acid substitutions, deletions, additions, or replacements.
  • variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids comprises a substitution group, and (d) variants in which the polypeptide or protein is fused to another polypeptide, such as serum albumin.
  • Conservative variants also refer to amino acid sequences having sequence changes that do not adversely affect the biological function of the protein.
  • a substitution, insertion, or deletion is described as adversely affecting the protein if the altered sequence disrupts or destroys the biological function associated with the protein.
  • the overall charge, structure, or hydrophobic-hydrophilicity of a protein can be altered without adversely affecting the biological activity.
  • the amino acid sequence can be altered to render the peptide more hydrophobic or more hydrophilic without adversely affecting the biological activity of the protein.
  • parent polypeptide As used herein, the terms "parent polypeptide”, “parent protein”, “precursor polypeptide” or “precursor protein” refer to an unmodified polypeptide that is later modified to form a variant.
  • the parent polypeptide can be a wild-type polypeptide, or a variant or engineered version of a wild-type polypeptide.
  • wild type or WT or “native” refers to an amino acid sequence found in nature, including allelic variants.
  • a wild type protein or polypeptide has an amino acid sequence that has not been intentionally modified.
  • a “variant" of an amino acid sequence includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • the term “variant” includes all splice variants, post-translationally modified variants, conformations, isoforms and species homologs, particularly those naturally expressed by a cell.
  • the term “variant” particularly includes fragments of an amino acid sequence.
  • Amino acid insertion variants comprise the insertion of one or more amino acids into a particular amino acid sequence.
  • amino acid sequence variants having insertions one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertions using appropriate screening of the resulting products are also possible.
  • Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein.
  • Amino acid deletion variants comprising deletions at the N-terminus and/or C-terminus of a protein are also referred to as N-terminal and/or C-terminal truncation variants.
  • Amino acid substitution variants are characterized by the deletion of one or more residues in the sequence and the insertion of another residue in its place. Preference is given to modifications located in an amino acid sequence that is not conserved between homologous proteins or peptides and/or to replacement of an amino acid with another amino acid having similar properties.
  • polypeptide according to the present invention may include conservative substitutions at positions other than the 2nd, 7th, 10th, and 13th amino acids of SEQ ID NO: 1.
  • a variant encompassed by the present invention is a protein comprising an amino acid sequence having 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 1 or 2, which maintains functional identity with a protein comprised of the amino acid sequence represented by SEQ ID NO: 1 or 2.
  • the amino acid changes in the peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids with a similar side chain.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-charged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids.
  • a conservative amino acid substitution comprises a substitution within the following groups:
  • valine isoleucine, leucine
  • Phenylalanine, tyrosine Phenylalanine, tyrosine.
  • a variant in one embodiment, includes conservative substitutions, wherein phenylalanine at position 1 in SEQ ID NO: 1 is replaced with tyrosine (F1Y); tyrosine at position 2 is replaced with phenylalanine (Y2F); alanine at position 3 is replaced with glycine (A3G); phenylalanine at position 5 is replaced with tyrosine (F5Y); alanine at position 8 is replaced with glycine (A8G); leucine at position 9 is replaced with valine (L9V) or isoleucine (L9I); and/or serine at position 10 is replaced with threonine (S10T).
  • acidic amino acid residue preferably relates to glutamic acid (glutamate, Glu) or aspartic acid (aspartate, Asp), particularly glutamic acid.
  • basic amino acid residue preferably relates to lysine (Lys) or arginine (Arg), particularly lysine.
  • the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of similarity or identity is preferably provided for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference amino acid sequence.
  • the degree of similarity or identity is preferably provided for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably contiguous amino acids.
  • the degree of similarity or identity is provided for the full length of the reference amino acid sequence. Alignments for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, e.g. Align, with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • Sequence similarity refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions.
  • Sequence identity refers to the percentage of amino acids that are identical between the sequences.
  • % identity is intended to refer to the percentage of amino acid residues that are identical between the two sequences being compared, obtained by best alignment, and this percentage is purely statistical, the differences between the two sequences being distributed randomly and over their entire length.
  • Sequence comparisons between two amino acid sequences are typically performed by optimally aligning those sequences and then comparing them, said comparison being performed by segments or "comparison windows" in order to identify and compare local regions of sequence similarity.
  • Optimal alignment of sequences for comparison may be performed manually, or by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol.
  • % Identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and then multiplying the result by 100 to obtain the % identity between the two sequences.
  • Homologous amino acid sequences according to the present specification exhibit at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98% or at least 99% identity with respect to amino acid residues.
  • amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. Manipulation of DNA sequences to produce peptides or proteins having substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, by solid phase synthesis and similar methods.
  • the fragment or variant of an amino acid sequence is preferably a "functional fragment” or "functional variant".
  • the term "functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which it is derived, i.e. is a functional equivalent.
  • the term “functional fragment” or “functional variant,” as used herein, refers to a variant molecule or sequence that comprises an amino acid sequence in which one or more amino acids have been altered, particularly compared to the amino acid sequence of the parent molecule or sequence, and which is still capable of performing one or more of the functions of the parent molecule or sequence, e.g., binding to a target molecule or contributing to binding to a target molecule.
  • the alteration in the amino acid sequence of the parent molecule or sequence does not significantly affect or alter the binding characteristics of the molecule or sequence.
  • the binding of the functional fragment or functional variant may be reduced but still significantly present, e.g., the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the binding of the functional fragment or functional variant may be increased relative to the parent molecule or sequence.
  • amino acid sequence "derived from" a specified amino acid sequence (peptide, protein or polypeptide) means the origin of the first amino acid sequence.
  • an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof.
  • An amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or a fragment thereof.
  • Rv0747/PE_PGRS10 suitable for use herein may be modified in sequence from a naturally occurring or native sequence derived therefrom, but which retains the desired activity of the native sequence.
  • isolated means altered or removed from its natural state.
  • a nucleic acid or peptide naturally occurring in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from its natural coexisting materials is “isolated.”
  • An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment, such as, for example, a host cell.
  • recombinant means “created through genetic manipulation.”
  • a “recombinant subject,” such as a recombinant cell, in the context of the present invention does not occur naturally.
  • naturally occurring means that a subject can be found in nature.
  • a peptide or nucleic acid is naturally occurring if it is present in an organism (including a virus), can be isolated from a source in nature, and has not been intentionally modified by humans in a laboratory.
  • the term “genetic modification” includes transfection of a cell with a nucleic acid.
  • the term “transfection” relates to the introduction of a nucleic acid, particularly RNA, into a cell.
  • the term “transfection” also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell.
  • RNA can be transfected into a cell and cause it to transiently express its encoded protein. Since the nucleic acid introduced during the transfection process is generally not integrated into the nuclear genome, the foreign nucleic acid will be diluted or degraded through mitosis.
  • Cells capable of episomal amplification of the nucleic acid significantly reduce the dilution rate. If the transfected nucleic acid is in fact retained in the cell and its progenitor cells, stable transfection must occur. Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.
  • cells genetically modified to express a receptor polypeptide and/or an antigen receptor are stably transfected with a nucleic acid encoding a receptor polypeptide and/or a nucleic acid encoding an antigen receptor, whereas typically, a nucleic acid encoding a ligand polypeptide and/or a nucleic acid encoding an antigen are transiently transfected into the cell.
  • nucleic acid refers to a polymer of the phosphate esters of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymine or deoxycytidine; "DNA molecule”), either in single-stranded form or as a double-stranded helix or any of their phosphate ester analogues such as phosphorothioates and thioesters.
  • Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and particularly DNA or RNA molecule, refer only to the primary and secondary structures of said molecules and are not limited to any particular tertiary configuration.
  • the term includes, among others, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and double-stranded DNA found as chromosomes.
  • the sequence may be described herein according to the general convention of presenting the sequence only in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand having the sequence matching the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation.
  • the DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.
  • the nucleic acid may be contained within a vector.
  • vector includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC).
  • BAC bacterial artificial chromosomes
  • YAC yeast artificial chromosomes
  • PAC P1 artificial chromosomes
  • Such vectors include expression as well as cloning vectors.
  • Expression vectors include plasmids as well as viral vectors, and generally contain the desired coding sequence and appropriate DNA sequences necessary for expression of the coding sequence operably linked in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system.
  • Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack functional sequences necessary for expression of the desired DNA fragment.
  • Viral vectors have been used for a wide range of gene transfer applications in living animals as well as cells.
  • Viral vectors that may be used include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, sendai virus, measles virus, simian virus 40, and Epstein-Barr virus vectors.
  • Non-viral vectors include plasmids, lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes.
  • the vector may include one or more regulatory regions and/or selectable markers that are useful for selecting, measuring, and monitoring the outcome of the nucleic acid delivery (e.g., delivery to tissues, persistence of expression, etc.).
  • expression vector refers to a vector, plasmid or vehicle designed to transform a host cell by expressing an inserted nucleic acid sequence.
  • the cloned gene i.e., the inserted nucleic acid sequence, is generally placed under the control of regulatory elements such as a promoter, a minimal promoter, an enhancer, and the like.
  • regulatory elements such as a promoter, a minimal promoter, an enhancer, and the like.
  • the initiation regulatory regions or promoters useful for directing expression of the nucleic acid in a desired host cell are numerous and are well known to those skilled in the art.
  • Virtually any promoter capable of directing expression of these genes includes, but is not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue-specific promoters, pathogenesis or disease-related promoters, developmental specific promoters, inducible promoters, light regulated promoters; SV40 early (SV40) promoter region, promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), E1A or major late promoter (MLP) of adenovirus (Ad), immediate early promoter of human cytomegalovirus (HCMV), herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GSPDH) promoter, phosphoglycer
  • Vectors can be transfected by methods known in the art, such as injection, transfection, electroporation, microinjection, transduction, cell fusion, lipofection, calcium phosphate precipitation (Graham, F.L. et al., Virology, 52:456(1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752(1987)), liposome-mediated transfection (Wong, T.K. et al., Gene, 10:87(1980); Nicolau and Sene, Biochim. Biophys.
  • the polynucleotides according to the present invention can be introduced in vivo by lipofection. Over the past several decades, there has been an increasing use of liposomes for in vitro nucleic acid encapsulation and transfection. Synthetic cationic lipids, which are designed to limit the difficulties and risks encountered with liposome-mediated transfection, can be used to prepare liposomes for in vivo gene transfection (Felgner et al., Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)).
  • cationic lipids can facilitate encapsulation of negatively charged nucleic acids, and can also facilitate fusion with negatively charged cell membranes (Felgner et al., Science 337:387 (1989)).
  • Particularly useful lipid compounds and compositions for the delivery of nucleic acids are described in WO95/18863, WO96/17823, and U.S. 5,459,127.
  • the use of lipofection to introduce exogenous genes into specific tissues in vivo has several practical advantages. Targeting of molecules to specific cells in liposomes presents one area of advantage. Direct transfection into specific cell types would clearly be particularly desirable in tissues with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids can be chemically linked to other molecules for targeting purposes (Mackey et al. 1988).
  • Targeted peptides such as hormones or neurotransmitters, and proteins, such as antibodies, or non-peptide molecules can be chemically bound to the liposomes.
  • cationic oligopeptides e.g., WO95/21931
  • peptides derived from DNA binding proteins e.g., WO96/25508
  • cationic polymers e.g., WO95/21931
  • nucleic acids described herein may be recombinant and/or isolated molecules.
  • transcription refers to the process by which the genetic code in a DNA sequence is transcribed into RNA.
  • the RNA can then be translated into peptides or proteins.
  • Encoding refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process, having a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties derived therefrom.
  • a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system.
  • Both the coding strand which is identical to the mRNA sequence and is usually provided as a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be said to encode a protein or other product of that gene or cDNA.
  • the present invention provides a host cell comprising a vector of the present invention.
  • the host cell includes prokaryotic (e.g., bacterial) and eukaryotic (e.g., fungal, yeast, animal, insect, plant) cells and may be any cell suitable for expression of a polypeptide according to the present invention.
  • Suitable prokaryotic host cells include, but are not limited to, E. coli (e.g., strains DH5, HB101, JM109 or W3110), Bacillus, Streptomyces, Salmonella, Serratia and Pseudomonas species.
  • Suitable eukaryotic host cells include, but are not limited to, COS, CHO, HepG-2, CV-1, LLCMK2, 3T3, HeLa, RPMI8226, 293, BHK-21, Sf9, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Aspergillus or Trichoderma species.
  • endogenous means any substance derived from or produced within an organism, cell, tissue, or system.
  • exogenous means any substance originating or produced outside an organism, cell, tissue, or system.
  • the term “expression” refers to the biological production of a product encoded by a coding sequence, and can be defined as the transcription and/or translation of a particular nucleotide sequence.
  • a DNA sequence comprising a coding sequence is transcribed to form messenger RNA (mRNA).
  • mRNA messenger RNA
  • the messenger RNA is then translated to form a polypeptide product having the relevant biological activity.
  • the expression process may also include additional processing steps for the RNA transcript product (e.g., splicing to remove introns), and/or post-translational processing of the polypeptide product.
  • the peptides, proteins, polypeptides, RNAs, RNA particles and additional agents, e.g., immune checkpoint inhibitors, described herein can be administered as pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment and can be administered in the form of any suitable pharmaceutical composition, which can include a pharmaceutically acceptable carrier and optionally can include one or more adjuvants, stabilizers, and the like.
  • the pharmaceutical composition is for use in therapeutic or prophylactic treatment, e.g., treating or preventing an inflammatory disease, such as those described herein.
  • composition relates to a formulation comprising a therapeutically effective substance, preferably together with a pharmaceutically acceptable carrier, diluent and/or excipient.
  • the pharmaceutical composition is useful for reducing the severity of, preventing or treating a disease or disorder by administering the pharmaceutical composition to a subject.
  • Pharmaceutical compositions are also known in the art as pharmaceutical dosage forms.
  • a pharmaceutical composition comprises a peptide, a protein, a polypeptide, an RNA, an RNA particle, an immune effector cell and/or an additional substance as described herein.
  • compositions of the present disclosure may include one or more adjuvants or may be administered together with one or more adjuvants.
  • adjuvant refers to a compound that prolongs, enhances, or accelerates an immune response.
  • adjuvants include heterogeneous compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., pertussis toxin), or immune-stimulating complexes.
  • adjuvants include, but are not limited to, cytokines such as LPS, GP96, CpG oligodeoxynucleotides, growth factors and monokines, lymphokines, interleukins, chemokines.
  • the cytokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFN ⁇ , IFN ⁇ , GM-CSF, LT-a.
  • adjuvants are aluminum hydroxide, Freund's adjuvant or oils such as Montanide®ISA51.
  • composition according to the present specification is generally applied as a “pharmaceutically effective amount” and as a “pharmaceutically acceptable formulation”.
  • pharmaceutically acceptable refers to the non-toxicity of a substance that does not interact with the action of the active ingredient of a pharmaceutical composition.
  • the term "pharmaceutically effective amount” or “therapeutically effective amount” means an amount that, alone or in combination with additional administration, achieves the desired response or desired effect.
  • the desired response preferably means inhibition of the progression of the disease. This includes slowing the progression of the disease, and in particular, stopping or reversing the progression of the disease.
  • the desired response may also be delaying the onset or preventing the onset of the disease or condition.
  • the effective amount of the compositions described herein will be determined based on the individual characteristics of the patient, including the condition being treated, the severity of the disease, age, physical condition, height and weight, the duration of the treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Accordingly, the dosage of the compositions described herein can be determined based on these various characteristics. If the patient does not respond sufficiently with the first administration, a higher dose (or a higher dose achieved by another, more local route of administration) may be used.
  • compositions of the present disclosure may include salts, buffers, preservatives, and optionally other therapeutic agents.
  • the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
  • Preservatives suitable for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens and thimerosal.
  • excipient refers to a material that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient.
  • excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.
  • diluting and/or thinning substance means a diluting and/or thinning substance.
  • the term “diluent” includes any one or more of a fluid, a liquid or solid suspension and/or a mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.
  • carrier refers to an ingredient which may be natural, synthetic, organic or inorganic, which is combined with an active ingredient to facilitate, enhance or enable administration of the pharmaceutical composition.
  • a carrier can be one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.
  • the pharmaceutical compositions herein comprise isotonic saline.
  • compositions may be selected depending on the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly.
  • the pharmaceutical compositions are formulated for topical administration or systemic administration.
  • Systemic administration can include enteral administration involving absorption through the gastrointestinal tract or parenteral administration.
  • parenteral administration means administration by any means other than via the gastrointestinal tract, such as intravenous injection.
  • the pharmaceutical compositions are formulated for systemic administration.
  • systemic administration is by intravenous administration.
  • the polypeptides described herein or variants thereof, or nucleic acid molecules encoding them are administered systemically.
  • co-administration means administering multiple compounds or compositions (e.g., immune effector cells, a polypeptide described herein or a variant thereof or a nucleic acid molecule encoding the same, and optionally an RNA encoding a vaccine antigen) to the same patient.
  • the multiple compounds or compositions can be administered simultaneously, essentially simultaneously, or sequentially.
  • the materials, compositions and methods described herein can be used to treat a subject having a disease, for example, a disease characterized by the presence of cells that express the disease antigen.
  • a disease for example, a disease characterized by the presence of cells that express the disease antigen.
  • Particularly preferred diseases are inflammatory diseases.
  • disease refers to an abnormal condition affecting the body of an individual.
  • a disease is often interpreted as a medical condition associated with specific symptoms and signs.
  • a disease may be caused by an agent originating from an external source, such as an infectious disease, or may be caused by an internal dysfunction, such as an autoimmune disease.
  • "disease” is used in a broader sense to refer to any condition that, when contacted by an individual, causes pain, dysfunction, distress, social problems, or death or similar problems to the individual suffering from the disease. In a broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and structural and functional atypical changes, which in different contexts and for different purposes may be considered distinct categories. Disease generally affects an individual not only physically but also emotionally, as living with various diseases can change the individual's outlook on life and personality.
  • the terms “treat,” “treating,” and “treatment,” and “prevent,” “preventing,” and “prevention” mean inducing a desired biological response, such as a therapeutic and preventive effect, respectively.
  • the therapeutic effect comprises one or more of: reducing/reducing the inflammatory disease, reducing/reducing the severity of the inflammatory disease (e.g., reducing or inhibiting the development of the inflammatory disease), reducing/reducing symptoms and inflammatory disease-related effects, delaying the onset of symptoms and inflammatory disease-related effects, reducing symptom severity of inflammatory disease-related effects, reducing the severity of acute episodes, reducing symptoms and inflammatory disease-related effects, reducing the latency of symptoms and inflammatory disease-related effects, improving symptoms and inflammatory disease-related effects, reducing secondary symptoms, reducing secondary infections, preventing relapse into the inflammatory disease, or frequency of relapse episodes, increasing the latency between symptom episodes, increasing the time to sustained progression, promoting remission, inducing remission, increasing remission,
  • Prophylactic effects may include complete or partial avoidance/inhibition or delay (e.g., complete or partial avoidance/inhibition or delay) of the development/progression of an inflammatory disease, and increased survival time of a subject following administration of the treatment protocol.
  • Treating an inflammatory disease includes treating a subject already diagnosed with any form of an inflammatory disease at any clinical stage or manifestation, delaying the onset or development or worsening or exacerbation of symptoms or signs of an inflammatory disease, and/or preventing and/or reducing the severity of an inflammatory disease.
  • terapéutica treatment means any treatment that improves the health status of a subject and/or prolongs (increases) the life span of a subject. Such treatment may be elimination of a disease in a subject, arrest or slowing of the progression of a disease in a subject, inhibition or slowing of the progression of a disease in a subject, reduction in the frequency or severity of symptoms in a subject, and/or reduction in relapse in a subject currently suffering from or previously suffering from a disease.
  • prophylactic treatment means any treatment intended to prevent a disease from occurring in a subject.
  • prophylactic treatment or “prophylactic treatment” are used interchangeably herein.
  • the terms “individual” and “subject” are used interchangeably herein. These terms refer to a human or other mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may be susceptible to or may be susceptible to a disease or disorder (e.g., an inflammatory disease), but may or may not have been susceptible to the disease or disorder.
  • the subject is a human.
  • the terms “individual” and “subject” do not imply a particular age, and thus encompass adults, elderly people, children, and newborns.
  • an "individual” or “individual” is a "patient.”
  • patient means an individual or entity in need of treatment, specifically an individual or entity suffering from a disease.
  • immune response refers to a coordinated body response to an antigen or cells expressing an antigen, and includes a cellular immune response and/or a humoral immune response.
  • cell-mediated immunity refers to a cellular response directed against cells characterized by the expression of an antigen, particularly by presentation of the antigen with class I or class II MHC.
  • the cellular response relates to cells called T cells or T lymphocytes, which act either as “helper” or “killer” cells.
  • Helper T cells also called CD4+ T cells
  • killer cells also called cytotoxic T cells, cytolytic T cells, CD8 + T cells, or CTLs kill cells that are involved in inflammatory diseases, thereby preventing the development of more diseased cells.
  • inducing [or inducing] an immune response may mean that there is no immune response to a particular antigen prior to induction, or it may mean that an immune response to a particular antigen exists at a baseline level prior to induction and is enhanced following induction.
  • inducing [or inducing] an immune response includes “potentiating [or enhancing] an immune response.”
  • immunotherapy refers to the treatment of a disease or condition by induction or enhancement of an immune response.
  • immunotherapy includes antigen immunization or antigen vaccination.
  • immuno or “vaccination” refers to the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.
  • macrophage refers to a subset of phagocytes produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products perform nonspecific phagocytosis, degrading pathogens by killing exogenous pathogens within the macrophage by hydrolytic and oxidative attack. Peptides derived from degraded proteins are presented on the cell surface of the macrophage, which can be recognized by T cells and can directly interact with antibodies on the surface of B cells, thereby activating T and B cells and further stimulating an immune response. Macrophages belong to the class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.
  • dendritic cell refers to another subset of phagocytes belonging to the antigen-presenting cell class.
  • dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are first transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells continuously sample their surrounding environment for pathogens such as viruses and bacteria. Once they come into contact with a presentable antigen, the cells are activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes.
  • Immature dendritic cells engulf pathogens, degrade their proteins into small fragments, and upon maturation present these fragments on the cell surface using MHC molecules.
  • these cells upregulate cell-surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, which greatly enhance their T cell activation potential. They also upregulate CCR7, a chemotactic receptor that causes dendritic cells to migrate to the spleen via the bloodstream or to lymph nodes via the lymphatic system.
  • they act as antigen-presenting cells, activating B cells by antigen presentation as well as helper T cells and killer T cells, in conjunction with non-antigen-specific co-stimulatory signals.
  • dendritic cells can actively induce T cell- or B cell-related immune responses.
  • the dendritic cells are splenic dendritic cells.
  • antigen presenting cell refers to a variety of cells capable of displaying, acquiring, and/or presenting at least one antigen or antigen fragment on (or at) their surface.
  • Antigen-presenting cells can be divided into professional antigen-presenting cells and non-professional antigen-presenting cells.
  • professional antigen-presenting cell is an antigen-presenting cell that constitutively expresses major histocompatibility complex class II (MHC class II) molecules required for interaction with na ⁇ ve T cells. If a T cell interacts with a complex of MHC class II molecules on the membrane of the antigen-presenting cell, the antigen-presenting cell produces co-stimulatory molecules that induce activation of the T cell.
  • Professional antigen-presenting cells include dendritic cells and macrophages.
  • non-professional antigen-presenting cell refers to an antigen-presenting cell that does not constitutively express MHC class II molecules upon stimulation by specific cytokines, such as interferon-gamma.
  • non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.
  • Antigen processing means the breakdown of an antigen into processing products, which are fragments of said antigen (e.g., breakdown of a protein into peptides) and combinations of one or more of these fragments with an MHC molecule (e.g., via binding) for presentation by a cell, such as an antigen presenting cell, to a particular T cell.
  • disease involving an antigen means any disease involving an antigen, for example a disease characterized by the presence of an antigen.
  • the disease involving an antigen may be an infectious disease or an inflammatory disease.
  • the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen.
  • the disease involving an antigen is a disease involving cells expressing the antigen, preferably on the surface of the cells.
  • infectious disease refers to any disease that can be transmitted from subject to subject or from organism to organism and is caused by a microbial agent (e.g., a cold). Infectious diseases are known in the art and include, for example, viral, bacterial or parasitic diseases caused by viruses, bacteria and parasites, respectively.
  • an infectious disease can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV/acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza and influenza.
  • inflammatory disease means a disease caused by inflammation in the body due to various causes, and in one embodiment of the present invention, the inflammatory disease is Alzheimer’s disease, atherosclerosis, asthma and allergic airway inflammation, gout, inflammatory bowel disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, myocardial infarction, multiple sclerosis or experimental autoimmune encephalitis, nephropathy, post-influenza hyperinflammation, graft-versus-host disease, stroke, silicosis, type 1 diabetes, obesity-induced inflammation or insulin resistance, rheumatoid arthritis, contact hypersensitivity, or traumatic brain injury.
  • Wild-type C57BL/6 mice were supplied by Samtako Bio Korea (Osan, Korea).
  • Primary bone marrow-derived macrophages (BMDMs) were collected from mice and cultured in DMEM with M-CSF (R&D Systems, 416-ML) for 3–5 days.
  • HEK293T cells ATCC-11268; American Type Culture Collection
  • DMEM Gibco
  • FBS FBS
  • nonessential amino acids amino acids
  • sodium pyruvate sodium pyruvate
  • streptomycin 100 ⁇ g/mL
  • penicillin 100 IU/mL
  • LPS Escherichia coli O111:B4, tlrl-eblps
  • ATP adenosine 5'-triphosphate
  • nigericin adenosine 5'-triphosphate
  • DSS dextran sulfate sodium
  • Actin (I-19) ASC (N-15-R), IL-18 (H-173-Y), caspase-1 p10 (M-20), HA (12CA5), Flag (D-8), GST (B-14), His (H-3), and V5 (C-9) were purchased from Santa Cruz Biotechnology.
  • IL-1 ⁇ and NLRP3 were from R&D system and Adipogen, respectively.
  • Wild-type NLRP3 (Int. J. Mol. Sci. 2020, 21, 8437) is 1036 amino acids long, ⁇ PYD was 93-1036 in wtNLRP3, ⁇ NACHT was 1-219 and 537-1036 in wtNLRP3, and ⁇ LRR was 1-741 in wtNLRP3. Plasmids for expressing full-length (FL) NLRP3 and mutants ( ⁇ PYD, ⁇ NACHT, and ⁇ LRR) were inserted into the pcDNA3 vector.
  • Plasmids encoding different regions of Rv0747 (1-801, 1-93, 94-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800) were amplified by PCR from FL Rv0747 cDNA and cloned downstream into a pEBG derivative encoding an N-terminal GST epitope tag flanked by BamHI and NotI sites.
  • Plasmids encoding different regions of ASC (1-195, 1-91, 107-195) were generated by PCR amplification from FL ASC cDNA and cloned downstream into a pEF derivative encoding a C-terminal Flag tag between the BamHI and NotI sites.
  • transient proteins encoded in the plasmids were produced in mammalian cells using the pEBG-GST mammalian fusion vector and the pEF-IRES-Puro expression vector. All constructs were verified to be 100% identical to the original sequence using an ABI PRISM 377 automated DNA sequencer.
  • NLRP3 or ASC peptides conjugated with 9R were provided by Peptron (Daejeon, Republic of Korea), manufactured and purified in acetate form to avoid undesirable intracellular reactions. Endotoxin levels were measured by Limulus amebocyte lysate test (Charles River Endosafe ®Endochrome-KTMUSA), and the concentrations of peptides used in the experiments were less than 3-5 pg/mL.
  • GST pull-down, western blot, and co-immunoprecipitation assays were performed with 293T and BMDM cells.
  • 293T cells were harvested and lysed in NP40 buffer containing a protease inhibitor cocktail (Roche, Basal, CH). After centrifugation, the supernatant was pretreated with protein A/G beads for 2 h at 4°C, and the pretreated lysate was combined with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences, Amersham, UK) and incubated for 4 h at 4°C. The precipitate was rinsed thoroughly with lysis buffer.
  • the proteins bound to the glutathione beads were eluted by boiling in sodium dodecyl sulfate (SDS) loading buffer for 5 min.
  • SDS sodium dodecyl sulfate
  • cells were harvested and lysed in NP40 buffer containing a protease inhibitor cocktail (Roche, Basal, CH).
  • NP40 buffer containing a protease inhibitor cocktail (Roche, Basal, CH).
  • Whole-cell lysates were pretreated with protein A/G agarose beads for 1 h at 4°C and then immunoprecipitated with the treated antibodies.
  • 1 mL of cell lysate was treated with 1–4 ⁇ g of antibody for 8–12 h at 4°C.
  • TNF- ⁇ , IL-6, IL-1 ⁇ , and IL-18 levels were measured in cell culture supernatants and mouse serum using the BD OptEIA ELISA system (BD Pharmingen). All assays were performed according to the manufacturer's instructions.
  • Rv0747 72 FYAQFVQALS 81 - NLRP3 interaction was monitored using a Fluormax-4 spectrofluorometer (HORIBA Scientific) as described previously.
  • Rv0747 72 FYAQFVQALS 81 or NLRP3 was labeled with BODIPY FL Iodoacetamide (ThermoFisher Scientific) according to the manufacturer's instructions.
  • Rv0747 72 FYAQFVQALS 81 or NLRP3 label was excited at 350 nm and detected through a cutoff filter at 512 nm.
  • Rv0747 72 FYAQFVQALS 81 or NLRP3 was designated as unlabeled NLRP3 or ASC for kinetic analysis.
  • the excitation and emission wavelengths used were 498 and 518 nm, respectively.
  • the acquired data were confirmed using the Grafit program, and all fluorescence measurements were performed at 25 °C in 30 mM Tris, pH 7.4, 150 mM NaCl, and 1 mM dithiothryitol.
  • the NLRP3 inflammasome complex plays an important role as a defense mechanism of host cells against pathogens.
  • recombinant NLRP3 rNLRP3-His
  • rNLRP3-His recombinant NLRP3
  • the purified rNLRP3-His complex was confirmed to bind to the Rv0747/PE_PGRS10 protein of Mycobacterium tuberculosis by silver staining and liquid chromatography-mass spectrometry (Fig. 1A).
  • NLRP3 protein is composed of PYD, NACHT, and LRR domains.
  • GST-tagged Rv0747 GST-tagged Rv0747 (GST-Rv0747) and Flag-tagged NLRP3 WT, PYD-deficient NLRP3 ( ⁇ NACHT), and LRR-deficient NLRP3 ( ⁇ LRR) were transfected into 293T cells, and a GST pull-down assay was performed.
  • Flag-NLRP3 WT, ⁇ PYD, and ⁇ NACHT were detected, but Flag-NLRP3 ⁇ LRR was not detected (Fig. 1B).
  • Rv0747/PE_PGRS10 is composed of PE and other domains, and the entire GST-Rv0747 domain (1-801), the PE domain (1-93), and the domains divided into about 100 amino acids each, and Flag-NLRP3 were used. Only the entire domain and the PE domain of Rv0747 were detected (Fig. 1C), and these results indicate that the PE domain and the LRR domain of NLRP3 interact with each other.
  • the protein sequence of the PE domain was analyzed using the PredictProtein tool, and it was confirmed that the 10 amino acid sequences from positions 72 to 81 would be important for the interaction with the NLRP3 protein (Fig. 1D).
  • BMDMs bone marrow-derived macrophages
  • inflammasomes secrete proinflammatory cytokines, interleukin (IL)-1 ⁇ /18.
  • LPS-stimulated BMDMs were treated with NLRP3 inflammasome activator (ATP, Nigericin, DSS, MSU, Alum), AIM2 inflammasome activator (poly(dA:dT)), NLRC4 inflammasome activator (flagellin), and NLRP1b inflammasome activator (LTx) and Rv0747-derived peptide
  • IL-1 ⁇ /18 was confirmed by ELISA and immunoblotting.
  • Flag-NLRP3-LRR When Flag-NLRP3-LRR was ubiquitinated and treated with Rv0747-derived peptide in a concentration-dependent manner, the ubiquitination of Flag-NLRP3-LRR did not show any change regardless of the peptide concentration (Fig. 3A). Similarly, the tyrosine phosphorylation form of Flag-NLRP3-LRR also did not show any change regardless of the peptide concentration (Fig. 3B).
  • Rv0747-derived peptide inhibits NLRP3 inflammasome activation through direct binding rather than modification of the LRR domain of NLRP3.
  • Short linear motifs such as peptides, can have their function increased or lost by one or two point mutations.
  • the middle QXVQ portion of the amino acid sequence of Rv0747 72 FYAQFVQALS 81 is symmetrical, so Q at position 75 was point-mutated to L, V at position 77 to H, and Q at position 78 to L.
  • Flag-NLRP3 and point-mutated GST-Rv0747 failed to bind to each other (Fig. 4), indicating that each amino acid may play an important role in the binding to NLRP3.

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Abstract

La présente invention concerne un polypeptide dérivé de mycobacterium tuberculosis ou un variant de celui-ci, et une composition pharmaceutique pour le traitement de maladies inflammatoires le comprenant. La composition pharmaceutique selon la présente invention peut être appliquée en tant qu'agent thérapeutique pour des maladies inflammatoires par liaison spécifique à NLRP3 et inhibition de l'activation de l'inflammasome NLRP3.
PCT/KR2024/003334 2023-06-01 2024-03-18 Composition pharmaceutique pour le traitement de maladies inflammatoires comprenant un polypeptide dérivé de mycobacterium tuberculosis Pending WO2024248287A1 (fr)

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