THERAPEUTICS FOR AUTOIMMUNE KIDNEY DISEASE: SYNTHETIC ANTIGENS
20220193210 · 2022-06-23
Inventors
Cpc classification
A61K47/61
HUMAN NECESSITIES
A61K47/6939
HUMAN NECESSITIES
A61K9/5161
HUMAN NECESSITIES
A61K47/542
HUMAN NECESSITIES
A61K47/549
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
International classification
Abstract
The present invention concerns therapeutics for autoimmune diseases and provides removal of inflammation-causing autoantibodies. In order to target the disease in the most efficient manner, a nanoconjugate complex is provided, comprising at least one specific antigen component recognized by autoantibodies related to the autoimmune disease, at least one helper moiety, and a nanoparticle carrier connecting the components. Each component of the therapeutic nanoconjugate complex has a specific function, yielding a nanoconjugate complex which facilitates specific binding, forming a stable antibody-therapeutic complex in the blood stream and rapid clearance of this complex to the liver.
Claims
1.-19. (canceled)
20. A nanoconjugate complex for treating an autoantibody-causing autoimmune disease, comprising the following components: i. at least one autoimmune disease-specific autoantigen recognized by autoantibodies related to said autoantibody-causing autoimmune disease, ii. at least one helper moiety selected from the group consisting of lipids, carbohydrates and polymers or combinations thereof, for providing functionalities to the complex including solubility, transport and clearing of said complex and autoantibody-autoantigen-nanoconjugate complexes, and iii. a nanocarrier connecting components i, ii,
21. The nanoconjugate complex according to claim 20 having one of the following general structures I: ##STR00010## wherein A is a nanocarrier; H, H1 and H2 are one or more different helper moieties selected from the group consisting of lipids, carbohydrates and polymers or combinations thereof for solubilizing, transporting and clearing autoantibody-autoantigen nanoconjugate complexes; D is one or more autoantibody-causing autoimmune disease-specific antigens; Ld and Lh are one or more different links or linkers in covalent or non-covalent binding; n.sub.h, n.sub.h1 and n.sub.h2 are the number of helper groups attacked to A; n.sub.h3 is the number of helper groups attacked to other helper groups; n.sub.d2 is the number of antigens groups attacked to A; and n.sub.d1 is the number of antigen groups attacked to a helper group.
22. The nanoconjugate complex according to claim 20, wherein A is selected from PAMAM, bis-MPA-azide dendrimer, chitosan, pullulan, silk fibroin, polyethyleneimine, poly(N-isopropylacrylamide) and poly(methacrylic acid), preferably a polysaccharide such as chitosan.
23. The nanoconjugate complex according to claim 22, wherein A is a polysaccharide, preferably chitosan or pullan, and the helper moieties (H, H1 and/or H2) are independently selected from lipid moieties and polymer moieties, such as hyaluronic acid and/or PEG, or combinations thereof.
24. The nanoconjugate complex according to claim 20 comprising the following components: i. at least one autoimmune disease-specific antigen recognized by autoantibodies related to an autoantibody-causing autoimmune disease, ii. at least one carbohydrate moiety, iii. at least one lipid moiety, iv. at least one polymer, and v. a nanocarrier connecting components i, ii, iii and iv.
25. The nanoconjugate complex according to claim 24 having the following general structure II: ##STR00011## wherein A is a nanocarrier to which n.sub.b lipid moieties (B), n.sub.c carbohydrate moieties (C), n.sub.d autoimmune disease-specific antigen moieties (D), and n.sub.e polymer moieties (E) are attached through direct links or linkers Lb, Lc, Ld, and Le, respectively; n.sub.d is at least 1 and n.sub.b, n.sub.e and n.sub.e are independent integers between 1 and N−3 and wherein the sum of n.sub.a+n.sub.e+n.sub.d+n.sub.e is between 4 and the total number of surface groups N available on A for covalent or non-covalent attachment.
26. The nanoconjugate complex according to claim 24, wherein A is selected from PAMAM, bis-MPA-azide dendrimer, chitosan, pullulan, silk fibroin, polyethyleneimine, poly(N-isopropylacrylamide) and poly(methacrylic acid), preferably a dendrimer such as PAMAM.
27. The nanoconjugate complex according to any claim 20, wherein the lipid(s) is/are the same or different fatty acid(s) selected from fatty acids containing straight or branched chains with a chain length 6 or more carbon atoms.
28. The nanoconjugate complex according to claim 27, wherein the lipid(s) is/are fatty acid(s) selected from caproic (hexanoic) acid, enanthic (heptanoic) acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid and palmitic acid.
29. The nanoconjugate complex according to claim 20, wherein the carbohydrate(s) is/are the same or different and selected from polysaccharides such as chitosan, hyaluronic acid and pullulan; or mono or disaccharides such as mannose, galactose, glucosamine, and their oligomers.
30. The nanoconjugate complex according to claim 20, wherein the polymer(s) is/are the same or different and selected from PEG, chitosan, pullulan and human serum albumin.
31. The nanoconjugate complex according to claim 20, wherein the antigen(s) is/are the same or different and selected from a peptide, phospholipid or an oligonucleotide related to the autoimmune disease.
32. The nanoconjugate complex according to claim 20, wherein the links or linkers connect by covalent or non-covalent binding the antigen and at least one helper moiety selected from the group consisting of lipids, carbohydrates and polymers or combinations thereof, to the carrier, and wherein the links or linkers are the same or different, consisting of one or more functional group(s) selected from ether, ester, disulfide, amide, 1,2,3-triazole, PEG, and electrostatic interaction.
33. The nanoconjugate complex according to claim 20, wherein the nanoconjugate complex has a size of about 100 to about 500 nm.
34. The nanoconjugate complex according to claim 20, wherein the autoimmune disease is selected from SLE-related diseases, CKD, RA, psoriasis, T1D, scleroderma and MS.
35. The nanoconjugate complex according to claim 20, wherein the antigen(s) is/are the same or different and selected from SEQ ID NO. 1-8 and 10.
36. A pharmaceutical composition comprising a nanoconjugate complex according to claim 20.
37. The nanoconjugate complex according to claim 20 for use in treating an autoimmune disease selected from a SLE-related disease, CKD, RA, psoriasis, T1D scleroderma and MS.
38. A method of preparing a nanoconjugate complex according to claim 24, comprising the steps: a. providing a backbone for use in connecting all the components of the nanoconjugate complex as set forth in steps b-e, b. linking at least one polymer component to the carrier c. linking at least one specific antigen component to the carrier d. linking at least one lipid component to the carrier e. linking at least one carbohydrate component to the carrier
39. A method of treatment of an autoimmune disease, comprising the steps: f. Providing a nanoconjugate complex according to a pharmaceutical composition according to claim 36; and g. Administering said nanoconjugate complex or said pharmaceutical composition to a patient suffering from autoimmune said disease.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0096] “Autoimmune disease” is a condition in which the immune system produces autoantibodies that instead of fighting infections, attack the body's own tissues.
[0097] The term “autoimmune kidney disease” as used herein means chronic kidney disease caused by autoantibodies
[0098] “CKD” means chronic kidney disease, which is a condition in which there is a progressive loss of kidney function.
[0099] “SLE” means systemic lupus erythematosus; which is an example of an autoimmune disease that may cause CKD.
[0100] “T1D” means type 1 diabetes, which is another example of an autoimmune disease that may cause CKD.
[0101] “RA” means rheumatoid arthritis
[0102] “MS” means Multiple Sclerosis
[0103] “Autoantibody” is an antibody produced by the immune system directed against the individual's own tissues.
[0104] “ANA” means anti-nuclear antibodies which are autoantibodies that bind to contents of the cell nucleus.
[0105] “Anti-dsDNA (a-ds-DNA) antibodies” are a group of ANA, the target antigen of which is double stranded DNA.
[0106] “Anti-histone antibodies” are autoantibodies that are a subset of ANA; they target protein components of nucleosomes, the DNA-protein complexes that form the substructure of transcriptionally inactive chromatin.
[0107] The term “nanoconjugate complex” (also just referred to as “nanoconjugates” or simply “conjugates”, or nanocarrier complex) as used herein, defines as a molecule comprising at least one specific antigen, at least one helper moiety, and a nanoparticle carrier. Such nanoconjugate complex may comprise (i) at least one antigen, (ii) at least one carbohydrate, (iii) at least one lipid, (iv) at least one polymer, and (v) a backbone connecting components i, ii, iii and iv. One nanoconjugate complex may comprise more than one of each of the antigen and helper moiety components if desired, the only limitation being the number of available surface groups/functional groups in the backbone for attachment of the components.
[0108] The term “backbone” as used herein, is a molecule that connects components of the nanoconjugate complex. It is also referred to as nanoparticle carrier or simply nanocarrier. The backbone functions as a carrier and transporter of the antigen or antigens in the cardiovascular system.
[0109] The term “helper moiety” broadly refers to molecules which help ensure the functionality of the nanoconjugate complex of clearing autoantibodies from the blood-stream, such as e.g. by contributing to solubility of the complex in the blood stream and ensuring the complex will not pass across the cell membranes.
[0110] The term “attachment site” as used herein, means sites on the backbone where the different components (antigen(s), carbohydrate(s), lipid(s), polymer(s)) of the nanoconjugate complex may be attached to the backbone by links or linkers.
[0111] “PAMAM” poly(amidoamine) is an example of a backbone component. It is a class of dendrimers made of repetitively branched subunits of amide and amine functionality. PAMAMs have a sphere-like shape overall, and are typified by an internal molecular architecture consisting of tree-like branching, with each outward ‘layer’, or generation, containing exponentially more branching points and possible functional groups.
[0112] The term “HSA” means human serum albumin, which is the serum albumin found in human blood.
[0113] “Antigen” is a structural molecule that binds specifically to an antibody. In the present invention, the antigens are recognized by autoantibodies, such as autoantibodies present in patients with SLE-related diseases, CKD, RA, psoriasis, T1D, scleroderma and MS. Antigens as used herein may be peptides, proteins, oligonucleotides, combinations and chemical analogues thereof.
[0114] The term “peptides” as used herein, means chains of amino acid monomers lined by peptide bonds with no distinct limitation on chain length.
[0115] The terms polypeptide and protein are used interchangeable herein.
[0116] The terms “polynucleotide” and “oligonucleotide” are used interchangeable herein, with no distinct limitation on chain length. Polynucleotide is a chain of nucleic acids, such as a DNA or RNA sequence.
[0117] The term “sequence identity” as used herein, indicates a quantitative measure of the degree of homology between two sequences of substantially equal length, such as two amino acid sequences or two nucleic acid sequences. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref−Ndif)100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson W. R and D. J. Lipman (1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). Alignment may be performed with sequence alignment methods such as ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.
[0118] The term “carbohydrate” as used herein, means a saccharide as well as saccharide derivatives such as amino sugars. The saccharide may be a mono-, di-, poly-, or oligosaccharide.
[0119] The term “lipid” as used herein, means fatty acid, a straight or branched aliphatic chain with no distinct limitation of number of carbon atoms.
[0120] In the present context the term “polymer” as a component of the nanoconjugate complex means a bulky molecule of a certain size that ensures stability of the nanoconjugate complex in biofluids as well as antigen representation to the autoantibodies.
[0121] “PEG” means polyethylene glycol, a polyether compound. PEGs are prepared by polymerization of ethylene oxide and comprise a wide range of molecules with the common formula C.sub.2nH.sub.4n+2O.sub.n+1, where n may range from 1 to 1000 or even greater.
[0122] The term “links” or “linkers” are used interchangeable herein, and indicates the connection between the backbone of the nanoconjugate complex and the antigen, carbohydrate, lipid and polymer components. More specifically, the linkers may comprise one or more functional group(s) selected from ether, ester, disulfide, amide, 1,2,3-triazole, or PEG. Covalent links made be formed by click chemistry.
[0123] Alternatively, the link may be noncovalent, such as an electrostatic interaction.
[0124] “Pyrogenicity” is the capacity to produce fever.
[0125] The present invention concerns therapeutics for autoimmune diseases caused by autoantibodies in a subject, such as a human or an animal such as a dog, a cat, a horse, etc. In order to target the disease in the most efficient manner, a multicomponent principle is applied. This means that each component of the therapeutic nanoconjugate complex disclosed in the present invention has a specific function.
[0126] The present invention concerns therapeutics for treatment of autoimmune diseases selected from different manifestations of SLE, including CKD caused by autoantibodies, alternatively referred to as autoimmune kidney disease in the present context, RA, T1D, Psoriasis, Sclerosis, Sjögren's Symptom, etc.
[0127] 1. A Nanoconjugate Complex
[0128] A first aspect of the present invention provides a nanoconjugate complex comprising the following components: [0129] i. at least one specific antigen recognized by autoantibodies related to an autoimmune disease, [0130] ii. at least one helper moiety, [0131] iii. a nanoparticle carrier connecting components I and ii.
[0132] In one embodiment of the aspect, the nanoconjugate complex comprises the following components: [0133] i. at least one specific antigen recognized by autoantibodies related to an autoimmune disease, [0134] ii. at least one carbohydrate, [0135] iii. at least one lipid, [0136] iv. at least one polymer, and [0137] v. a backbone connecting components i, ii, iii and iv.
[0138] The at least one of components (i), (ii), (iii) and (iv) means at least one of these helper moieties per carrier A. If a helper moiety is present on a carrier in more than one copy, all copies may be the same or different. The different helper moieties are preferably present on a carrier independently in between 4 and 20 copies
[0139] The novel nanoconjugate complex contains at least one specific antigen for the disease-causing antibodies, which binds to the inflammatory antibodies and blocks their further biological activity. The nanoconjugate complex additionally has helper components/moieties that aid rapid clearance of the antigen-antibody complex from the blood stream, whereby further inflammation development is prevented, a solubilizing enhancer, a bulky group and a backbone holding it all together and function as a carries of cargo.
[0140] In one preferred embodiment, the nanoconjugate complex has the following general structure II:
##STR00005##
[0141] wherein A is a nanopolymeric carrier to which n.sub.b lipid moieties (B), n.sub.c carbohydrate moieties (C), n.sub.d disease-specific antigen moieties (D), and n.sub.e polymer moieties (E) are attached through direct links or linkers Lb, Lc, Ld, and Le, respectively; n.sub.d is at least 1 and n.sub.b, n.sub.c and n.sub.e are independent integers between 1 and X−3 and wherein the sum of n.sub.a+n.sub.c+n.sub.d+n.sub.e is between 4 and the total number of surface groups X available on A for covalent or non-covalent attachment.
[0142] The nanoconjugate complex of this aspect of the present invention consists of a complex of different functionalities B, C, D and E collected on the surface of the carrier A which ensures that the nanoconjugate complex is soluble in the blood stream, large enough for not passing cell membranes, presents at least one antigen (in a protected way), is able to selectively bind circulating autoantibodies, is tolerable (non-toxic and non-immunogenic) to the subject/patient and is able to transport, removed and deplete the autoantibody-nanoconjugate complex from the blood-stream in the subject/patient.
[0143] In a preferred embodiment, A is a synthetic polymer, such as PAMAM, PNIMAM etc.
[0144] The nanoconjugate complex may comprise at least one B per backbone (A), i.e. one or more B per A. If B is present on A in more than one copy, all copies of B may be the same or different. The nanoconjugate complex may comprise at least one C per A, i.e. one or more C per A. If C is present on A in more than one copy, all copies of C may be the same or different. The nanoconjugate complex may comprise at least one D per A, i.e. one or more D per A. If D is present on A in more than one copy, all copies of D may be the same or different. The nanoconjugate complex may comprise at least one E per A, i.e. one or more E per A. If E is present on A in more than one copy, all copies of E may preferably be the same. The numbers of B, C, D, and E on A are mutual independent. The number of B, C, D, and E is only limited by the number of “available attachment sites” on A, as described in greater detail in the following section concerning the nanocarrier of the nanoconjugate complex.
[0145] In an alternative embodiment, B may be coupled to C and/or D and/or E; C may be coupled to B and/or D and/or E; D may be coupled to B and/or C and/or E; and E may be coupled to B and/or C and/or D; and linked to A.
[0146] The location on the backbone of the different components of the nanoconjugate complex with respect to one another may be any physically/chemically possible constellation and should not be limited to the layout illustrated in the general structures above.
[0147] 1.1 Backbone of the Nanoconjugate Complex, the Nanoparticle Carrier
[0148] The backbone of the nanoconjugate complex is a molecule that connects all the components of the complex. Depending on the choice of backbone, the number of available attachment sites for the components may differ.
[0149] Different dendrimers may be engineered as candidates for therapeutic application. Dendrimers are repetitively branched molecules which are typically symmetric around the core, and often adopt a spherical three-dimensional morphology. One example is Bis-MPA azide dendrimer, a hyperbranched nanoparticle based on the 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) monomer unit. The azide architecture of this dendrimer can easily be functionalized using click chemistry, which is a well-known method for the synthesis of dendrimers. Applying Bis-MPA azide dendrimer as backbone in the nanoconjugate complexes of the present invention, the azide-branches represent available attachment sites for the antigen(s), carbohydrate(s), lipid(s), and polymer(s) components.
[0150] Another dendrimer: poly amido amide (PAMAM) dendrimer has large number of amino and carboxyl groups which may represent available attachment sites for the antigen(s), carbohydrate(s), lipid(s), and polymer(s) components of the nanoconjugate complex of the present invention. PAMAMs have a sphere-like shape overall, and are typified by an internal molecular architecture consisting of tree-like branching, with each outward ‘layer’, or generation, containing exponentially more branching points. As shown in
[0151] It is crucial that the backbone does not induce toxicity. Based on the literature [Ayatollahi S, et al. Int J Biochem Cell Biol. 2017 November; 92:210-217], PAMAM and similar scaffold molecules are safe in terms of toxicity and can be applied as backbone for the nanoconjugate complex of the present invention. Other useful backbone units will be apparent for the skilled person.
[0152] In one embodiment, the backbone of the nanoconjugate complex is a dendrimer, such as a scaffold molecule selected from PAMAM or bis-MPA-azide dendrimer of any generation size. In another embodiment, the backbone of the nanoconjugate complex is selected from a carbohydrate such as chitosan and pullulan, or a biomolecule such as silk fibroin, or polyethyleneimine, poly(N-isopropylacrylamide), and poly(methacrylic acid). In a preferred embodiment, the backbone of the nanoconjugate complex is PAMAM.
[0153] Based on the structure of the backbone, multiple sites may available for the components of the nanoconjugate complex of the present invention to attach to. All available attachment sites on the backbone of the nanoconjugate complex may or may not have functional components attached, such as a specific antigen (D), a lipid (C), a carbohydrate (A), and a polymer component (E) attached. For example, for G5-PAMAM, it may be that only 25-30% of all the termini (branches) are modified with a functional component. However, less termini branches (active sites) may be modified, such as about 5%, about 10%, about 15% or about 20%; or more termini branches are modified, such as about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and up to 100%. Structure I should therefore be regarded merely as an illustration that the nanoconjugate complex comprises a specific antigen (D), a lipid (C), a carbohydrate (A), and a polymer component (E), but not be regarded as limited to one of each component. The nanoconjugate complex may comprise one or more of each component. The ratio of the components is not restricted to 1:1, but may vary. The different components may be mixed and attached randomly throughout the branched backbone; or their location may specifically be preselected.
[0154] In another preferred embodiment or the present invention, the nanoconjugate complex has one of the following general structures I:
##STR00006##
[0155] wherein A is nanocarrier, such as a polysaccharide or polypeptide; H, H1 and H2 are one or more different helper moieties; D is one or more autoimmune disease-specific antigens; Ld and Lh are one or more different links or linkers in covalent or non-covalent binding; n.sub.h, n.sub.h1 and n.sub.h2 are the number of helper groups attacked to A; n.sub.h3 is the number of helper groups attacked to other helper groups; n.sub.d2 is the number of antigens groups attacked to A; and n.sub.d1 is the number of antigen groups attacked to a helper group.
[0156] When the carrier A is selected from one of the polysaccharides known for such purposes, such as chitosan or pullulan, it is not necessary to include carbohydrates as helper moiety. In order to create a nanoparticle of the right size for not penetrating cell membranes and for securing solubility in the blood stream, helper moieties such as hyaluronic acid may be conjugated to a chitosan core. The chitosan and/or the helper moiety, e.g. hyaluronic acid, may be further decorated with surface neutralizing helper moieties, such as for example PEG. Chitosan, hyaluronic acid and PEG are all known not to be toxic or immunogenic, and thus relatively safe for use in medical treatment. The antigen can be attached to the carries, e.g. chitosan, or one of the helper moieties, e.g. hyaluronic acid or PEG. All moieties may be conjugated by covalent or non-covalent binding.
[0157] 1.2 Specific Antigen Component of the Nanoconjugate Complex
[0158] The autoimmune disease specificities of the nanoconjugate complexes are limited to the conjugated antigens. All helper moiety selected from carbohydrates, lipids, polymers as well as the carries are not disease specific, but contribute to the complex by adding further beneficial properties as described. The specific antigen is recognized by autoantibodies related to autoimmune disease; it thereby binds and helps facilitate clearance of the autoimmune disease autoantibodies. The patterns in individual patients vary; in other words the same antigens get recognized but at a different level across antigens for each patient. Selection of disease specific antigen sequences may be done by traditional antigen library screening, or more time and cost efficient by rational design, using a combination of computational and laboratory screening, supported by studying available literature. A successful disease specific antigen is stable, with a high affinity for the disease associated autoantibody in the patient.
[0159] The specific antigen of the nanoconjugate complex of the present invention, recognized by autoantibodies related to an autoimmune disease, may be a nucleic acid sequence, a peptide, a phospholipid or other cell-related components. The nanoconjugate complex comprises at least one specific antigen, i.e. one or more specific antigen(s). If the specific antigen is present in more than one copy, all copies may be the same or different. If the specific antigens are a combination of different antigens within the same nanoconjugate complex, such a combination may be of different oligonucleotides within the same nanoconjugate complex, different peptides within the same nanoconjugate complex, or a mixture of oligonucleotide(s) and a peptide(s) within the same nanoconjugate complex.
[0160] In one embodiment, the specific antigen(s) is/are the same or difference and selected from peptide(s) and oligonucleotide(s) related to autoimmune kidney disease. In another embodiment, the specific antigen(s) is/are the same or difference and selected from peptide(s) and oligonucleotide(s) related to RA.
[0161] Another important factor in selecting the right helper moieties and way of conjugating the autoantigen or component mimicking a specific disease-related autoantigen is the need of quenching any immunogenic epitopes of the antigen, such that it will not come in contact with immune cells which would potentially light a new immune reaction. The binding and properties of the helper moieties results in such epitopes being “buried” in the conjugate and thus quenched for connecting to cell membranes. The autoantibody-specific epitopes only gets into contact with circulating autoantibodies.
[0162] Peptides related to autoimmune kidney disease may be selected from peptides mimicking histone H3 peptides owing to confirmed efficacy of ANA binding, such as SEQ ID NO. 3. Further, for reducing potential toxicity and cost of a therapeutic, a part of the original sequence may be used, such as SEQ ID NO. 5 and SEQ ID NO. 6, which are derived from SEQ ID NO. 3. Further, clearance of autoantibodies-nanoconjugate complexes may be improved by liver targeting peptides, such as SEQ ID NO. 4.
[0163] Oligonucleotides related to autoimmune kidney disease may be selected from DNA sequences target by anti-DNA antibodies in SLE disease. SEQ ID NO. 1 and SEQ ID NO. 2 are examples of such oligonucleotides. Other examples are SEQ ID NO. 7 and SEQ ID NO. 8, where SEQ ID NO. 8 is anti-SLE specific.
[0164] In a further embodiment, the autoimmune kidney disease specific antigen is characterized by being recognized by autoantibodies related to autoimmune kidney disease and is selected from oligonucleotides SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 8 as well as oligonucleotides with >60% sequence identity to SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 8; in another embodiment, the autoimmune kidney disease specific antigen is selected from peptides SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 as well as peptides with >60% sequence identity to SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6; in yet another embodiment, the autoimmune kidney disease specific antigen is a combination of two of more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 8 as well as sequences with >60% sequence identity to any of the selected SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 8.
[0165] In a further embodiment, the autoimmune kidney disease specific antigen is characterized by being recognized by autoantibodies related to autoimmune kidney disease and is selected from oligonucleotides SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 8 as well as oligonucleotides with >80% sequence identity to SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 8; in another embodiment, the autoimmune kidney disease specific antigen is selected from peptides SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 as well as peptides with >80% sequence identity to SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6; in yet another embodiment, the autoimmune kidney disease specific antigen is a combination of two of more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 8 as well as sequences with >80% sequence identity to any of the selected SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 8.
[0166] In a further embodiment, the autoimmune kidney disease specific antigen is characterized by being recognized by autoantibodies related to autoimmune kidney disease and is selected from oligonucleotides SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 8 as well as oligonucleotides with greater than 82, 84, 86, 88, 90, 92, 94, 96, or 98% sequence identity to SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 8; in yet another embodiment, the autoimmune kidney disease specific antigen is selected from peptides SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 as well as peptides with greater than 82, 84, 86, 88, 90, 92, 94, 96, or 98% sequence identity to SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6; in another embodiment, the autoimmune kidney disease specific antigen is a combination of two of more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 8 as well as sequences with greater than 82, 84, 86, 88, 90, 92, 94, 96, or 98% sequence identity to any of the selected SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 8.
[0167] In a preferred embodiment, the autoimmune kidney disease specific antigen of the nanoconjugate complex is oligonucleotide SEQ ID NO. 1 or any oligonucleotides with >80% sequence identity to SEQ ID NO. 1. In a more preferred embodiment, the autoimmune kidney disease specific antigen of the nanoconjugate complex is oligonucleotide SEQ ID NO. 1.
[0168] In a preferred embodiment, the autoimmune kidney disease specific antigen of the nanoconjugate complex is oligonucleotide SEQ ID NO. 1 or any oligonucleotides with greater than 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98% sequence identity to SEQ ID NO. 1. In a more preferred embodiment, the autoimmune kidney disease specific antigen of the nanoconjugate complex is oligonucleotide SEQ ID NO. 1.
[0169] Differences within the antigen sequences between different patients are one reason for differences in sequence identity as discussed above. Another reason is the possibility of changes in the antigen leading to the same or enhanced recognition and/or binding to the autoantibodies.
[0170] RA and certain forms of psoriasis are known to be related to the presence of citrullinated proteins or peptide in affected patients. Specific citrullinated peptide epitopes can be selected by screening of protein fragments and their mutated variants in for example RA sera. As an example, a library of 25 citrullinated peptide epitopes derived from fibrinogen, vimentin and histone 3 were screened against sera from RA patients and one of these peptides were found to bind RA sera selectively. Having selected the most potent peptide epitope, it was included into nanoparticles loaded for evaluation by a series of in vitro assays. The library screened comprised the citrullinated peptides SEQ ID NO. 9 to SEQ ID NO. 33. SEQ ID NO. 10 has been shown to comprise a RA-autoantibody-specific antigen epitope.
[0171] In a preferred embodiment, the autoimmune RA specific antigen of the nanoconjugate complex is peptide SEQ ID NO. 10 or any peptide with >80% sequence identity to SEQ ID NO. 10. In a more preferred embodiment, the autoimmune RA specific antigen of the nanoconjugate complex is peptide SEQ ID NO. 10 or any oligonucleotides with greater than 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98% sequence identity to SEQ ID NO. 10. In a most preferred embodiment, the autoimmune RA specific antigen of the nanoconjugate complex is peptide SEQ ID NO. 10.
[0172] 1.3 Lipid Component of a Nanoconjugate Complex with Structure II
[0173] Lipids influence the transport, biodistribution, efficacy and cellular uptake of different drugs; hence lipids can facilitate increased solubility and adsorption as well as enhanced bioavailability. The lipid component of the nanoconjugate complex acts as a clearance signal for the antibody:nanoconjugate complex [Hutchinson et al, Pept Sci. 2017 February; 23(2):82-94]. This is not limited to a certain fatty acid, however longer chains (C7 and greater) are known to target the molecules to the liver and enhance digestion. Moreover, together with PEG (see below), the lipid component improves biodistribution and prolongs half-life in serum.
[0174] The nanoconjugate complex comprises at least one lipid, i.e. one or more lipid(s). If the lipid is present in more than one copy, all copies may be the same or different.
[0175] In one embodiment, the lipid component of the nanoconjugate complex is one or more fatty acid(s), selected from the natural aliphatic fatty acids such as those readily available from commercial suppliers. The fatty acids may be straight chain or branched; they may be saturated, unsaturated or a combination hereof. In a preferred embodiment, the fatty acids of the nanoconjugate complex of the present invention are unbranched and saturated. In a preferred embodiment the lipid component of the nanoconjugate complex is selected from enanthic (heptanoic) acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid and palmitic acid.
[0176] In another embodiment, the lipid component may be a combination of different lipids within the same nanoconjugate complex, such as a combination of two or more different fatty acids within the same nanoconjugate complex, such as where the fatty acids are selected from caproic (hexanoic) acid, enanthic (heptanoic) acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid and palmitic acid.
[0177] 1.4 Carbohydrate Component of a Nanoconjugate Complex with Structure II
[0178] The carbohydrate component of the nanoconjugate complex increases the solubility of the lipidated molecule. Simultaneously a carbohydrate might promote clearance of the inflammation-causing dead cells and their parts (called microparticles) as well as apoptotic bodies. Recently, it has been shown that microparticles are being extensively secreted to the blood of patients having autoantibody-related kidney disease [Giannella et al. Cardiovasc Diabetol. 2017; 16: 118]. These particles contain surface proteins that recognize specific carbohydrates. A carbohydrate component is therefore included in the nanoconjugate complex of the present invention to help clear these.
[0179] The nanoconjugate complex comprises at least one carbohydrate, i.e. one or more carbohydrate(s). If the carbohydrate is present in more than one copy, all copies may be the same or different.
[0180] In one embodiment the carbohydrate component of the nanoconjugate complex is selected from the available literature on microparticle surface glycosylation. Glucosamine is a prominent precursor in the biochemical synthesis of glycosylated proteins and lipids. Other carbohydrates related to microparticle surface glycosylation comprise D-mannose, D-galactose and their oligomers. Diverse carbohydrates can be applied depending on the overall conjugate design. The carbohydrates may be a mono-, di-, poly-, or oligosaccharide.
[0181] In one embodiment the carbohydrate component of the nanoconjugate complex is selected from mannose, galactose, glucosamine, and their oligomers. In a preferred embodiment the carbohydrate component of the nanoconjugate complex is selected from galactose and glucosamine.
[0182] In a further embodiment, the carbohydrate component may be a combination of different carbohydrates within the same nanoconjugate complex, such as a combination of two or more different carbohydrates selected from mannose, galactose, glucosamine, and their oligomers.
[0183] 1.5 Polymer Component of a Nanoconjugate Complex with Structure II
[0184] The nanoconjugate complex comprises a polymer component to ensure the stability in biofluids and antigen representation to the autoantibody (IgG, IgA or IgM). A polymer such as PEG can, by increasing the molecular weight of a molecule, impart several significant pharmacological advantages, such as improved drug solubility, extended circulating life, increased drug stability, and enhanced protection from proteolytic degradation. Therefore the polymer needs to be hydrophilic. With regard to the size, the polymer can be a broad range, such as starting with PEG3000 and going up to PEG20000. PEGylation thereby aids in the effective delivery of the nanoconjugate complex to the targeted destination. Human serum albumin (HSA) is another option to achieve these beneficial properties.
[0185] The nanoconjugate complex comprises at least one polymer, i.e. one or more polymer(s). If the polymer is present in more than one copy, all copies may be the same or different.
[0186] In one embodiment, the polymer component of the nanoconjugate complex may be selected from functionalized carbohydrates such as chitosan and pullulan, or protein derivatives that are known to improve biodistribution of biological drugs such as human serum albumin. In a preferred embodiment, the polymer of the nanoconjugate complex is PEG. PEG is commercially available in different forms and can be selected in combination with the carrier and other helper moiety properties of the nanoconjugate complex.
[0187] In another embodiment, the polymer component may be a combination of different polymers within the same nanoconjugate complex, such as a combination of two or more polymers selected from PEG, chitosan, pullulan and human serum albumin.
[0188] 1.6 Links or Linkers of the Nanoconjugate Complex
[0189] The links or linkers Lb, Lc, Ld, Le, in Structure II connect the antigen, carbohydrate, lipid and polymer components to the backbone and Ld and Lh in structure I connect the antigen and helper moiety to the backbone. The selection of conjugation chemistry depends on the chemical properties of the starting material and the desired stability of the bond created in the product. The links or linkers may be the same or different. In a preferred embodiment, the linkers may be any functional group such as ether, ester, disulfide, amide, 1,2,3-triazole, or PEG. Alternatively, the link may be noncovalent, such as an electrostatic interaction.
[0190] In a further embodiment, the linkers may comprise a combination of two or more functional groups within one linker, the functional groups being selected from ether, ester, disulfide, amide, 1,2,3-triazole, and PEG.
[0191] In a selected embodiment, the link is non-covalent.
[0192] 2 Preparation of Nanoconjugate Complexes
[0193] A second aspect of the invention relates to a method for preparing nanoconjugate complexes of the present invention.
[0194] In one embodiment, the nanoconjugate complex of structure I of the present invention is prepared by a method comprising the steps: [0195] a. providing a nanocarrier for use in connecting all the components of the nanoconjugate complex [0196] b. linking at least one helper moiety to the carrier [0197] c. linking at least one specific antigen to the carrier or the helper moiety wherein step b and c may be carried out in any order or be combined.
[0198] In another embodiment, the nanoconjugate complex of structure II of the present invention is prepared by a method comprising the steps: [0199] a. providing a nanocarrier for use in connecting all the components of the nanoconjugate complex as set forth in steps b-e, [0200] b. linking at least one polymer component to the carrier [0201] c. linking at least one specific antigen component to the carrier [0202] d. linking at least one lipid component to the carrier [0203] e. linking at least one carbohydrate component to the carrier [0204] wherein two or more of the steps b, c, d, and e may be combined; and the steps may be carried out in any chosen order
[0205] In any embodiment of preparing nanoconjugates, covalent binding or non-covalent binding may be chosen as desired. For covalent binding, click chemistry is the preferred synthesis and well known in the art. Additional dialysis and labelling steps may further be introduced where needed, as identified by a person skilled in the art.
[0206] With regard to the structure of the assembly, the different components may be linked randomly to the carrier backbone, or the location may be preselected. Further, multiple units of each component may be linked to the backbone of the nanoconjugate complex, such that the final nanoconjugate complex comprises one or more of each component. There is no defined restriction on the ratio of the components. The available functional surface groups on the carrier define to upper limit of the total number of the components. Preferably between 10 and 70% of the available surface groups are occupied by the antigen(s) and the helper moieties. More preferred, between 20 and 50% of the groups are occupied.
[0207] The antigen and helper moieties, such as carbohydrate, lipid, and polymer components, may be linked to the backbone of the nanoconjugate complex by covalent attachment, such as through linkers or links as specified below; or may be linked by noncovalent attachment. In a preferred embodiment, the linkers may be any functional group such as ether, ester, disulfide, amide, 1,2,3-triazole, or PEG. Alternatively, the link may be noncovalent such as an electrostatic interaction. In further embodiment, the linkers may comprise a combination of two or more functional groups within one linker, the functional groups being selected from ether, ester, disulfide, amide, 1,2,3-triazole, and PEG. Depending on the type of link or linker, different attachment protocols known by a person skilled within the art may be used to connect the different components of the nanoconjugate complex, such as including but not limited to standard PEGylation, click chemistry attachment, and NHS (N-hydroxysuccinimide) chemistry attachment protocols.
[0208] In one embodiment, the nanoconjugate complex is PEGylated. PEGylation is the process of attaching strands of the polymer PEG to molecules, thereby producing alterations in the physiochemical properties including changes in conformation, electrostatic binding, hydrophobicity etc. PEGylation may be performed according to standard protocols known by a person skilled in the art, such as done by hydroxysuccinimide chemistry [Alibolandi et al. 2017. Int J Pharm 519, 352-364].
[0209] In one embodiment, one or more selected component(s) of the nanoconjugate complex is linked to the backbone by noncovalent attachment by slowly adding the component(s) in a preselected ratio to a stirred solution containing the backbone and let the mixture incubate for a sufficient time period.
[0210] In another embodiment, one or more selected component(s) of the nanoconjugate complex is linked to the backbone by click chemistry [WO2007011967A2]. The reaction may be performed according to standard protocols known by a person skilled in the art, such as done by the classic copper-catalyzed click reaction of an azide and an alkyne [Development and Applications of Click Chemistry. Gregory C. Patton. Nov. 8, 2004]. In a preferred embodiment, the pH may vary from acidic to basic, but concentrations of the reaction components shall be kept in a low milimolar range.
[0211] In another embodiment, a selected component of the nanoconjugate complex is linked to the backbone by NHS (N-HydroxySuccinimide) ester reaction with free amino groups. Amino groups are nearly always contained in proteins and peptides, modification of these biopolymers by NHS ester reaction is therefore especially common. Other examples are amino-oligonucleotides, amino-modified DNA, and amino-containing sugars. The reaction may be performed according to standard protocols known by a person skilled in the art. The reaction of NHS esters with amines is strongly pH-dependent: at low pH, the amino group is protonated, and no modification takes place. At higher-than-optimal pH, hydrolysis of NHS ester is quick, and modification yield diminishes. In a preferred embodiment, pH value for NHS (N-hydroxysuccinimide) ester reaction is 8.3-8.5.
[0212] Compared to the standard multi-step synthesis of low molecule therapeutic drugs, the preparation of the nanoconjugate complex of the present invention is experimentally simple as is evident from the above description as well as example 1. The synthesis scheme is flexible and can be adjusted for the specific nanoconjugate composition, aiming at the most efficient representation of the antigen within the product.
[0213] 3. Treating Autoimmune Diseases with Nanoconjugate Complexes
[0214] A third aspect of the invention relates to a pharmaceutical composition comprising the nanoconjugate complex. The therapeutic nanoconjugate complex may be of the general structure I or II, or may comprise a combination of two or more nanoconjugate complexes, such as complexes comprising different specific antigens, different carbohydrates, different lipids, or even different polymers. For example, the pharmaceutical combination comprises two different complexes, wherein the antigen is different, such as two different oligonucleotides, two different peptides or a combination of oligonucleotide(s) and peptide(s). In the same way the pharmaceutical combination may comprise three or even more different nanoconjugate complexes.
[0215] The nanoconjugate complex may be part of a pharmaceutical composition further comprising existing low molecular drugs and biologics (for example methotrexate and/or a monoclonal antibody such as Rituximab [Cravedi. G Ital Nefrol. 2012 May-June; 29(3):274-82; discussion 292]).
[0216] Important requirements for therapeutic drugs include low toxicity, high target binding specificity, and prolonged effect in vivo. These properties are obtained in the nanoconjugate complex of the present invention by combining multiple active components within one complex: active antigen, solubilizing reagents, several state-of-the-art helper molecules that aid sufficient biodistribution and clearance from the blood stream when the target antibody is recognized and bound. Further, most of the components of the nanoconjugate complex of the present inventions are biomolecules; this ensures low toxicity of the therapeutic product.
[0217] A fourth aspect of the invention relates to using the nanoconjugate complex in treating autoimmune diseases, such as autoimmune kidney disease, RA, psoriasis, T1D, sclerosis and others, and provides a method of treatment comprising the steps: [0218] a. providing at least one nanoconjugate complex or a pharmaceutical composition according to the invention; and [0219] b. administering said nanoconjugate complex(es) or said pharmaceutical composition to a patient suffering from an autoimmune disease.
[0220] Patients to be treated with the nanoconjugate complex may be humans or animals suffering from CKD, caused by autoantibodies, RA or other autoimmune diseases at any disease stage.
[0221] The nanoconjugate complex may be administered to the patient by intravenous injection, transfusion, intramuscular injection, or by other such methods known by a person skilled in the art for administering pharmaceutical complexes. The nanoconjugate complex may be administered in several dosages with a selected interval for a selected period of time. The use of therapeutic may be adjusted based on measurements of autoantibody levels in the blood. It is most preferred to administer the nanoconjugates directly to the blood stream by iv administration.
[0222] The therapeutic nanoconjugate complex of the present invention addresses the cause of kidney disease, RA and other auto immune diseases and is in that way safer and more efficient than currently used symptomatic drugs. The nanoconjugate complex not only binds the autoantibodies but also helps clear them from the blood stream such that new inflammation is hindered. The autoantibodies do therefore not accumulate in the body, and further success of the treatment does not rely on in vivo degradation of the autoantibodies. Using this nanomaterial, the autoimmune diseases can be treated earlier in its course and with a better outcome for the patient since the tissue damage by chronic inflammation is prevented.
EXAMPLES
[0223] The following examples are merely intended to illustrate the principle of the present invention and therefore in no way intended to limit the scope of the claimed invention.
Example 1: In Vitro Assay—Identification of Suitable SLE/CKD Antigens
[0224] The suitability of different possible antigens aiming at autoantibodies involved in kidney autoimmune disease (Table 1) was tested prior to synthesizing nanoconjugate complexes. Oligonucleotides relating to autoimmune kidney disease were selected from DNA sequences targeted by anti-DNA antibodies in SLE disease. TCCTTTCTTTCTTTCTT (SEQ ID NO. 1) and (TTAGGGTTAGGGTTAGGGTTAGGGTTAG) SEQ ID NO. 2 were selected for testing such oligonucleotides. One tested peptide, ARTKQTARKSTGGKAPGGC (SEQ ID NO. 3) relates to autoimmune kidney disease mimicking histone H3 peptides owing to a confirmed efficacy of ANA binding. Parts of the original sequence, ARTKQTAR (SEQ ID NO. 5) and KQTARKSTGGKAPG (SEQ ID NO. 6), derived from SEQ ID NO. 3 are also tested.
TABLE-US-00001 TABLE 1 Selected antigens aiming at kidney disease. Component Antigen Sequence D1 Oligonucleotide: 5′ TCCTTTCTTTCTTTCTT 3′ (SEQ ID NO. 1) D2 G-quadruplex oligonucleotide: 5′ TTAGGGTTAGGGTTAGGGTTAGGGTTAG 3′ (SEQ ID NO. 2) D3 Histone peptide: ARTKQTARKSTGGKAPGGC (SEQ ID NO. 3) D4 P41 peptide: SWLRRIWRWICKVLSRFK (SEQ ID NO. 4) D5 Histone peptide H3s1: Ac-ARTKQTAR (SEQ ID NO. 5) D6 Histone peptide H3s2: Ac-KQTARKSTGGKAPG (SEQ ID NO. 6)
[0225] SEQ ID NO. 4 is a liver targeting peptide which when attached to the carries may be used to improve the clearance of the autoantibodies-nanoconjugate complexes.
[0226] Binding of antigens shown in Tables 1 to SLE/CKD disease stated sera was confirmed by enzyme linked immunosorbent assay (ELISA). Maxisorb 96 well plates (NUNC Thermofisher, Germany) were coated with individual antigens at concentration 5 μg/mL in 1×PBS overnight (room temperature; 150 μl/well). After washing with 1×PT (2×300 μl/well, PT: 50 μl Tween-20 in 1 L 1×PBS), the plates were blocked with 1×PTB (1 h, 37° C.; 100 μl/well, PTB: 20 g BSA, 50 μl Tween-20 in 1 L 1×PBS). Incubation with SLE/CKD plasma at desired dilution was performed at 37° C. for 1.5 h using diluent: 2 g BSA, 50 μl Tween-20 in 1 L 1×PBS (100 μl/well). This was followed by washing (2×300 μl 1×PBS) and incubation with HPR-labelled secondary antibody for 1.5 h at 37° C. using same diluent and dilution of the secondary antibody provided by supplier (HPR-conjugated a-aIgG or a-aIgM; Sigma). Subsequent washing (2×300 μl PT) and incubation with freshly prepared TMB-H.sub.2O.sub.2 solution (Sigma; 100 μl/well) was followed by adding a stop solution (1M H2SO4; 50 μl/well) and reading resulting absorbance values at 450 nm on Magellan Tecan microplate reader.
[0227] Linear range for each antigen (D1, D2, D3 and D4) was determined via testing series of control dilutions (control sera purchased from Immunovision in dilutions 1:50 to 1:2000). The linearity confirmed that the selected concentration range was suitable for the detection of antibodies, and that other sera/assay components did not interfere with the result. According to the results plasma dilutions 1:100-1:500 were within linear range of the assay for each antigen (R.sup.2>0.95).
Example 2: Synthesis of Nanoconjugate Complexes
[0228] Different nanoconjugate complexes aiming at kidney autoimmune disease were prepared as described below.
[0229] 2.1 Composition of the Synthesized Nanoconjugate Complexes
[0230] The synthesized nanoconjugates complexes comply with the general Structure II:
##STR00007##
[0231] wherein A is a nanoparticle backbone/carrier to which at least one (n.sub.b) lipid (B), at least one (n.sub.c) carbohydrate (C), at least one kidney autoimmune disease specific antigen (n.sub.d) (D), and at least one (n.sub.e) polymer (E) are attached through links or linkers Lb, Lc, Ld, and Le, respectively.
[0232] The compositions of each of the synthesized nanoconjugate complexes are summarized in Table 2 with the different components further specified in Table 3.
TABLE-US-00002 TABLE 2 Composition of synthesized nanoconjugate complexes for treatment of kidney disease (No. 1-5, and 7-8) and controls (No. 6, 9 and 10) Conjugate no. Composition 1 A + B1 + C1 + D1 + E 2 A + B2 + C1 + D1 + E 3 A + B2 + C2 + D1 + E 4 A + B1 + C1 + D4 + E 5 A + B1 + C2 + D4 + E 6 A + C1 + D1 + E 7 A + B1 + C1 + D5 + E 8 A + B1 + C1 + D6 + E 9 A + C1 + D5 + E 10 A + C1 + D6 + E
TABLE-US-00003 TABLE 3 Specification of the components of the nanoconjugate complexes Components A PAMAM polymer (G5) B1 Heptanoic acid B2 Pentadecanoic acid C1 Galactose C2 Glucosamine D1 Oligonucleotide: 5′ TCCTTTCTTTCTTTCTT 3′ (SEQ ID NO. 1) D2 G-quadruplex oligonucleotide: 5′ TTAGGGTTAGGGTTAGGGTTAGGGTTAG 3′ (SEQ ID NO. 2) D3 Histone peptide: ARTKQTARKSTGGKAPGGC (SEQ ID NO. 3) D4 P41 peptide: SWLRRIWRWICKVLSRFK (SEQ ID NO. 4) D5 Histone peptide H3s1: (acetylated)-ARTKQTAR (SEQ ID NO. 5) D6 Histone peptide H3s2: (acetylated)-KQTARKSTGGKAPG (SEQ ID NO. 6) E PEG3000
[0233] In
[0234] All the reagents and buffers used in the preparation of the nanoconjugate complexes are listed in Table 4. Reagents and buffers obtained from commercial suppliers were used as received.
TABLE-US-00004 TABLE 4 Used reagents and buffers Reagent/buffer Origin D-(+)-Galactose G0750 Sigma-Aldrich Denmark D-Glucosamine CDS021691 Aldrich Denmark PAMAM Ethylenediamine core, generation 536709 Aldrich USA 5.0 Heptanoic acid 75190 Sigma Denmark Pentadecanoic acid W433400 Sigma Denmark DNA Oligonucleotide: 5′ custom ordered from IDT, Belgium TCCTTTCTTTCTTTCTT 3′ G-quadruplex forming DNA oligonucleotide: custom ordered from IDT, Belgium 5′ TTAGGGTTAGGGTTAGGGTTAGGGTTAG 3′ Peptide: ARTKQTARKSTGGKAPGGC custom ordered from Caslo lab, Denmark Peptide: SWLRRIWRWICKVLSRFK custom ordered from Caslo lab, Denmark Peptide: (acetylated) ARTKQTAR Made in-house at DTU Chemistry, Denmark Peptide: (acetylated) KQTARKSTGGKAPG Made in-house at DTU Chemistry, Denmark PEG3000 Sigma Aldrich Denmark 81230 PBS buffer tablets Sigma Denmark P4417 MQ water: prepared by deionizer (Milipore) (DTU Chemistry, Denmark) in house Sodium bicarbonate Sigma Denmark S5761 10K Dialysis kit Thermo fisher Germany 88404
[0235] The following plastics and other minor equipment was used:
[0236] Microcentrifuge tubes (Thermo Germany, 2150N), glass vials (VWR Denmark, 113459), pipetman set (Gilson, Inc, UK, PIPETMAN® Classic), pipet tips (Gilson, Inc, UK, PIPETMAN DIAMOND Tips—Sterilized Filter Tips, 14324), shaker (Eppendorf Innova® S44i Shaker, USA), centrifuge (Thermo fisher Germany, R0165).
[0237] 2.3 Synthesis of PEGylated PAMAM Precursor
[0238] The amounts of different components to be added are reported in Table 5 and were calculated as follows:
[0239] Amount of PAMAM dendrimer=20 mg
[0240] The ratio of PEG3000:Dendrimer=1:3.33
[0241] Amount of PEG3000 needed=(30/100)×20=6 mg
[0242] The ratio of PEG with NHS and EDC is 1:8:8, giving masse ratio 6 mg: 48 mg:48 mg
TABLE-US-00005 TABLE 5 Amounts of different components for synthesis of PEGylated PAMAM precursor Materials CAS no MW State Amount NHS N-hydroxysuccimide 6066-82-6 115.09 powder 48 mg EDC 1-ethyl 3-(3-dimethyl amino- 25952-53-8 119.70 powder 48 mg propyl)carbodiimide PEG3000 PEG polymer(MAL-PEG-COOH) 948595-08-2 MP 3000 6 mg G5 PAMAM Dendrimer G5 163442-68-0 5912.32 liquid 20 mg
[0243] The synthesis of PEGylated PAMAM precursor was performed by the following steps [Alibolandi et al, Int J Pharm. 2017 Mar. 15; 519(1-2):352-364]: [0244] 1. PAMAM dendrimer was dissolved in PBS at pH 7.4 [0245] 2. 6 mg PEG-COOH was added to the solution and mixed with NHS and EDC in the ratio of MAL-PEG-COOH:NHS:EDC 1:8:8 [0246] 3. The mixture was stirred for 16 hours at 800 rpm, protected from light. [0247] 4. Dialysis was done (cut off: 14000 Da) against 3 mL of PBS pH 7.4 for 24 hours to remove unconjugated PEG and residual EDC/NHS. [0248] 5. SPEED VAC was used to reduce sample volume [0249] 6. Characterization was done by DLS and 1H-NMR.
[0250] NMR was used to characterize the conjugates upon the selected conjugation and after the purification by a 24 h long dialysis with a 10-20 MWKO membrane. The observed change in NMR signal confirmed the successful attachment of antigen in the preparation of the nanoconjugate complex.
[0251] 2.4 Attachment of Antigen/Carbohydrate/Lipid to PEGylated PAMAM Precursor
[0252] The different components of the nanoconjugate complex may be attached to the backbone by different methods. A generalized description of different “attachment methods” is provided below as well as the step by step process for the synthesis of nanoconjugate complex no 1.
[0253] General noncovalent attachment protocol: In order to yield the target nanoconjugate in 1×PBS (1 mL), 10% excess of the required amount of components was added dropwise over 2 hours to a stirred solution of PEGylated dendrimer in 1×PBS (100 mM, pH 7.2, 2 mL). The reaction mixture was stirred for 24 hours and afterwards analyzed by 1H-NMR on Bruker 400 (DTU Chemistry, Denmark). The product has been concentrated using 10K dialysis kit from Thermo Fisher Scientific.
[0254] General click chemistry attachment protocol: A 10% excess of azide or alkyne reagents has been added to PEGylated PAMAM containing corresponding alkyne or azide groups in 100 mM TEAA buffer at pH 7.0 (2 mM solution PAMAM in 1 mL). Alkyne/azide containing PAMAM precursors are available from commercial supplies such as Sigma, or can be made together with attaching PEG using NHS-alkyne and NHS-azide reagents available from e.g. Lumiprobe (see example for nanoconjugate 1). The components that get clicked such as a peptide or a DNA sequence are obtained from commercial suppliers or synthesized in house, with including the desired alkyne or azide label for click chemistry. Afterwards copper-THPTA and freshly prepared ascorbic acid were added, and the resulting mixture was degassed by argon and kept at room temperature for 12 h. The resulting mixture was subjected to dialysis through 10K device (Thermo Fisher Scientific). The product was analyzed by 1H-NMR on Bruker 400 (DTU Chemistry).
[0255] General NHS chemistry attachment protocol: A 10% excess of NHS reagents has been added to PEGylated PAMAM containing free amino groups in 100 mM bicarbonate buffer pH 8.3 (2 mM solution PAMAM in 1 mL). The reaction was gently stirred at room temperature for 4 h and then subjected to dialysis through 10K device (Thermo Fisher Scientific). The product was analyzed by 1H-NMR on Bruker 400 (DTU Chemistry).
[0256] 2.5 Step-by-Step Protocol for Synthesis of Nanoconjugate Complex No 1
[0257] The General Synthesis Strategy for Conjugate 1:
[0258] 1. NHS-PEG coupling to G5 PAMAM, dialysis;
[0259] 2. Coupling reaction with 3 equivalent heptanoic acid, dialysis;
[0260] 3. Treatment of product of step (2) with Azide-PEG3-amine (Limiprobe, cat no. 218-1g);
[0261] 4. Click chemistry of galactose-alkyne and oligonucleotide-alkyne mixture to the product of step (3) in a molar ratio 3:1.
[0262] Step 1. Same as described in section 1.3 “Synthesis of PEGylated PAMAM precursor”, followed by dialysis using 10 kDa MWKO membrane (Thermo Fisher Scientific, cat no 87729) following the manufacturer's protocol.
[0263] Step 2. PEGylated G5 PAMAM prepared in step 1 was re-suspended in 100 mM PBS (pH 7.2), at concentration 1 mg/mL (1 mL). Heptanoic acid (6 μL of 10 mM stock in t-BuOH), and EDC (12 μL of 10 mM stock in DMFA) were added, and the reaction was kept at room temperature for 36 hours, at gentle stirring (200 rpm). The product was dialyzed using 14 kDa MWKO membrane (Thermo Fisher Scientific) for 24 h and restored in 1 mL 100 mM bicarbonate buffer, pH 8.2, for the step 3.
[0264] Step 3. A solution of step 2 product in 100 mM bicarbonate (pH 8.2) was incubated at room temperature for 2 h with N,N′-diisopropylcarbodiimide (7 μL; DIC, Sigma D125407) and N-hydroxysuccinimide (10 μL of 10 mM stock in milliQ water; Sigma (cat no 130672). Azide-PEG3-amine (15 μL of 10 mM stock in milliQ water; Lumiprobe, cat no. 218-1g) was added, and the reaction was kept at room temperature for 12 hrs at gentle stirring (200 rpm). The product was dialyzed using 14 kDa MWKO membrane (Thermo Fisher Scientific) for 24 h and restored in 200 μL 100 mM TEAA buffer, pH 7.2
[0265] Step 4. To a solution of step 3 product in 100 mM TEAA buffer (pH 7.2; 30 μL at concentration 1 mg/mL), the following reagents were subsequently added: DMSO (20 μL), D1-5′-hexynyl oligonucleotide (4.4 nmol hexynyl/TCCTTTCTTTCTTTCTT in 5 μL milliQ water; IDT), beta-Gal-TEG-Alkyne (8.8 nmol in 5 μL milliQ water; IDT, IRIS BIOTECH GBB1385), copper TBTA ligand (10 uL, 10 mM stock, Lumiprobe 21050) and freshly prepared ascorbic acid (5 μL of 25 mM stock in milliQ water; Sigma A92902-25G). The mixture was degassed by flushing with argon over 3 min and kept at room temperature on 200 rpm shaking for 48 hr. The product was dialyzed using 20 kDa MWKO membrane (Thermo Fisher Scientific) for 24 h and restored in 200 μL 100 mM PBS, pH 7.2.
Example 3: Solubility of Nanoconjugate Complex
[0266] The effect of a carbohydrate component on solubility of the nanoconjugate complex is demonstrated by a titration experiment, where an increasing amount of glycose is coupled to G5-PAMAM-PEG-butyric acid. 100 mM PBS buffer (1 mL, pH 7.2, Sigma) was added dropwise to the evaporated conjugate (1 mg). The solubility can be measured simply by filtering, drying and weighing the undissolved conjugate.
Example 4: Pyrogenicity and Complement Activation
[0267] To evaluate pyrogenicity and complement activation that can interfere with the in vivo testing of the nanoconjugate complexes, standard procedures were used [Huang et al. Osteoarthritis Cartilage. 2016 October; 24(10): 1769-1775]. Pyrogenicity is most often caused by bacterial antigens such as lipopolysaccharide (LPS). Pyrogenicity was tested for the nanoconjugate complexes vs. commercial LPS standard in dilutions 1:100 down to 1:100,000, in fresh MQ water and laminar setting. The result confirms the absence of any contamination in the conjugates as all plates were “clean” for the conjugates of the present invention, while gelation was observed in the presence of LPS as a control (
Example 5: Stability of Nanoconjugate Complexes: Oxidation, Storage and Aggregation
[0268] The solution of a nanoconjugate or a control in 100 mM PBS (pH 7.2, Sigma), at concentration 1 mg/mL was stored at −20° C. or +4° C. Aliquots were taken every month. To evaluate for aggregation, supernatant samples were analyzed by measuring optical density at 260 (DNA antigens)/280 nm (peptide antigens). A decrease in optical density >15% was considered as an aggregation. Oxidation was tested by HRMS, comparing the mass of initial compound to the sample. Increased mass by m/z 32 and more confirmed the oxidation. Chemical composition was tested in LC MS, elution system isocratic gradient tBuOH in PBS buffer 10->90%, flow speed 1 ml/min, on C18 analytical column, connected to the MS spectrometer. The mass of a sample was compared to the initial compound used as a control. Deviation in the LC profile and MS >15% was considered a decomposition. The results are reported in Table 6.
TABLE-US-00006 TABLE 4 Stability studies of nanoconjugates 1-5 to oxidation and storage analyzed by HRMS and to aggregation studied by LC MS. Oxidation Storage Storage Aggregation Compound stability −20° C., at −20° C., at +4° C., −20/+4° C., no. months months months % 1 >12 >12 >12 <5/<5 2 8 >12 4 7/<5 3 7 >12 6 11/<5 4 >12 7 4 25/14 5 >12 8 4 30/22
Example 6: Toxicity Study
[0269] All the antigens and nanoconjugates selected by rational design have been tested in terms of cellular toxicity; this includes all the nanoconjugates shown in Table 2. Conjugates 1-5 were tested using IL-19 and KIM-1 biomarkers in cell lines and in vivo. Conjugates 6-10 were tested in human blood using viability assay, see below.
[0270] Apparent toxicity is sequence dependent and requires careful design and testing of the selected antigens and helper molecules. Cell line tests were performed to ensure ethically reasonable transfer of the conjugate from bench to animal model. BHK cells were selected due to robustness and low cost. BHK (baby hamster kidney) cells (BHK-21 [C-13] ATCC® CCL-10™, USA) were grown in MEM medium (BioWhittaker, USA). Complete medium for BHK cells is MEM+2 mM L-glutamine (Sigma Denmark, 1294808); +5% fetal bovine serum (Sigma F2442). Cells were grown in a humidified, 37° C., 5% CO2 incubator and split three times at 1:5, reaching 90% confluency. Cell growing took 11 days in total. A solution of nanoconjugate complex at concentration 1 nM or 10 nM was added to cells in 1×PBS and incubated for 24 h. Afterwards the cells were fixed with MeOH (Sigma, cell culture grade), crashed and subjected to analysis of IL-19 using commercial ELISA kits (The Quantikine human IL 19 kit, R&D Products, USA), following manufacturers protocols. The results are shown in Table 7.
TABLE-US-00007 TABLE 5 Toxicity study of nanoconjugate in BHK cell line and in vivo (NZB/W, IV administration, 10 nM; blood sample analyzed 36 h after initial administration). BHK: IL-19, NZB/W: IL-19, NZB/W: KIM-1, Compound pg/mL pg/mL ng/mL no. (cell lysate) (plasma) (plasma) 1 88 76 1.4 2 75 95 2.2 3 94 104 3.1 4 122 170 2.4 5 134 211 5.2 Negative control* na 73 1.4 Gentamicin treatment na 78 2.05 *Healthy mice; na = not applied
[0271] As it is shown in Table 7, no apparent toxicity was detected in the analysis using BHK cells, measuring the levels of IL-19 which is a biomarker for toxicity. When synthetic peptide TAT (positive control for toxicity in cellular assays) was added, the IL-19 levels did increase (data not shown). Since all levels were within the normal range (70-150 μg/mL), confirming no apparent toxicity of the conjugates, the conjugates were then tested in the NZB/W mice.
[0272] Nine-week-old NZB/W mice were kindly provided by Heegaard group, Statens Serum Institute, Denmark; ten mice (all females) were kept in sterile boxes covered by a filter and fed sterile water and food. The mice were grown for 10 weeks and reached weight 17-19 g in average. The mice were bled before the experiment to check for the presence of anti-dsDNA antibodies (a-dsDNA) by standard ELISA. Only those with a-dsDNA in titer 1:1000-1:12000 were used for this study. The nanoconjugate complexes were added to the tail vein. Nanoconjugate complex was administrated using IV in 1×PBS, applying the nanoconjugate complex at 160 μg/kg animal weight for 10 nM concentration. Blood samples were withdrawn 36 hours after initial administration and subjected to analysis of IL-19 and KIM-1 using commercial ELISA kits (The Quantikine human IL 19 kit, R&D Products, USA; KIM 1 ELISA kit ADI-900-226-0001, ENZO Life Sciences, USA), following manufacturers protocols. These results are also shown in Table 5.
[0273] Based on the stability and toxicity studies, nanoconjugate complex no 1 was selected as the most potent candidate and studied further in vivo.
Example 7: In Vivo Mice Assay
[0274] For the further in vivo test, a well-described mice model was selected: NZB/W mice, these have been used as a model for autoimmune disease since the early 1960s. Mice of this hybrid cross develop an autoimmune disease resembling human SLE.
[0275] Nine-week-old NZB/W mice were kindly provided by Heegaard group, Statens Serum Institute, Denmark; they were grown and tested for the presence of anti-dsDNA antibodies as described in example 6. Nanoconjugate complex was added to the tail vein, administrated using IV in 1×PBS, applying the nanoconjugate complex at 160 μg/kg animal weight for 10 nM concentration, and 16 μg/kg animal weight for 1 nM concentration. Two mice were used for each conjugate. Mice received IV conjugate/control administration (same amount each time), with 12 h intervals for 5 days, and afterwards giving the same dose with 3 day interval over 3 weeks. Treatment with Gentamicin was used in control animals of same strain and age, at dosage 10 mg/kg animal weight and 1 mg/kg for final 10 nM and 1 nM administration, respectively. Administration regimen for same as for nanoconjugate complexes: IV in tail vein with 12 h interval for 5 days, and afterwards giving the same dose with 3 day interval.
[0276] Blood samples were withdrawn at time points: 24 h, 48 h, 1 week, 4 weeks after the beginning of each treatment. Plasma was centrifuged using Qiagen blood storage tubes and stored at −20° C. prior to analyses. ELISA analysis was performed using manufacturer's protocols in sera dilution 1:100 to 1:500. The results for nanoconjugate complex 1 are presented in
[0277] Since there are no spikes in the disease activity index that could be caused by complement activation, the data in
Example 8: Interaction of Nanoconjugate Complexes with Human Primary Blood Cells
[0278] Cytotoxicity and uptake by blood cells are all potential issues for the nanoconjugates. This was studied by FACS (fluorescence-activated cell sorting) using primary human blood cells and conjugates 1 and 6-10 (table 2), along with a G5 PAMAM control. To run FACS, the nanoconjugate complexes and the control were additionally labelled with CY5.5 NHS reagent (Lumiprobe), following this protocol: Conjugates 1,6-10 at concentration 1 mg/mL in 100 μL bicarbonate (0.1 M, pH 8.3) were added to 20 μl 10 mM dye stock in the DMSO. The mixture was stirred at 300 rpm in dark overnight and afterwards dialyzed at 10 KDa MWKO (Thermofisher dialysis cassette Cat no 87729), following the manufacturer's procedure. The conjugate was kept in 100 mM PBS at pH 7.2 afterwards.
[0279] For FACS experiments, fresh whole blood from five donors (Stanford University Hospital) was used. The protocol for the blood work up and incubation with conjugates is given below. [0280] 1. Pool together the blood from the 2 heparin tubes (total˜20 ml) [0281] 2. Add 20 ml commercially available RPMI buffer (no FBS) (Sigma R0883) [0282] 3. 1600 rpm, 5 min, discard top pink layer [0283] 4. Repeat step 2-3 twice [0284] 5. Aliquot 250 ul of blood to each FACS tube. [0285] 6. Lyse with 3 ml of ACK lysis buffer (Gibco #A10492-01) for 10 mins, RT. [0286] 7. 1600 rpm, 5 min, discard supernatant [0287] 8. Wash twice with 2 ml RPMI buffer. [0288] 9. Resuspend with 250 ul RPMI buffer [0289] 10. Count cells. Take 5 ul of cells and add 95 ul Trypan blue. [0290] 11. Add 250 ul of designated conjugate prepared in RPMI buffer to each of the tubes (250 ul of 20 nM) [0291] 12. Mix cells with nanotubes by vortexing three times 5 counts each. [0292] 13. Incubate for 30 mins in 37° C. Caps are kept loose to keep cells alive. [0293] 14. Stop incubation by transferring tubes to ice for 20 min [0294] 15. Add 2 ml RPMI buffer, 1600 rpm, 5 min, discard supernatant [0295] 16. Repeat step 12. [0296] 17. Resuspend cell pellet in 100 ul of milliQ aqua solution (LD aqua diluted 1:1000 PBS), 10 minutes at room temperature, covered with foil. [0297] 18. Wash with FACS buffer, (for washes if using BD FACS tubes use 500 ul for each wash) Spin 5 minutes, 1500 rpm and remove supernatant. FACS buffer was 2% calf serum (Sigma 12133C), 1 mM EDTA, 0.1% sodium azide. [0298] 19. Resuspend pellet in 100 ul blocking buffer* (5% heat inactivated AB serum and 5% goat serum in PBS (Sigma P4417))) [0299] 20. Incubate on ice for 15 min [0300] 21. Add antibody CD20 (CD20 antibody (0.N.85): sc-70582, Santa Cruz Biotechnology) and HLADR (Anti-HLA-DR antibodies, human (clone: AC122), Miltenyl Biotec) directly to cells (2 ul for each antibody/100 ul of cell suspension). [0301] 22. Incubate on ice, 30 min [0302] 23. spin 1500 rpm 5 min 4° C. [0303] 24. Fix cells by re-suspending pellet in 200 ul of BD cytofix solution (BD 554714). Add Cytofix solution slowly to the cell pellet while vortexing or with frequent vortexing. Incubate at RT in the dark for 20-30 mins. [0304] 25. Wash with 500 ul of FACs buffer and re-suspend in 200 ul of FACS buffer. [0305] 26. Keep at 4 C, avoid light till analysis (within 24 h)
[0306] The resulting samples were analyzed on BD FACS instrument (BD FACSLyric™). The results for specific cellular uptake are given in Table 8.
TABLE-US-00008 TABLE 6 Fluorescence intensity in cell population for each nanoconjugate complex and controls Conjugate T cell B cell monocytes NK Neutrophils G5 (control) 11 21 5 24 35 1 8 10 6 16 20 6 5 12 11 18 37 7 14 32 30 21 12 8 11 20 35 24 8 9 14 12 30 21 60 10 11 18 35 24 76 Cy5.5-RNA negative 2 1 1 4 2 control
[0307] Table 8 shows that PEGylated G5 PAMAM dendrimer (control) is being mostly taken up by neutrophils, and less by NK, T, and B cells, while monocytes take up only a little of G5. When the oligonucleotide, carbohydrate and lipid are added (conjugate 1), levels for all cells are somewhat similar to G5 alone, whereas removing the lipid part (conjugate 6) increases the uptake by neutrophils. For histone peptides (conjugates 7-10) the effect of lipid becomes dramatic. The uptake is high when the lipid is absent (conjugates 9-10), by neutrophils mostly, and it drops down to 8-12 in presence of the lipid (conjugates 7-8).
[0308] This data suggests three important facts: (1) T cells, B cells and monocytes are little affected by the conjugates. This means no activation and confirms low cytotoxicity/side effects for the therapeutics; (2) Peptide-containing therapeutics have higher uptake by monocytes than DNA conjugates, however T and B cells are still little affected; (3) Lipidation can be used as an instrument to fine tune the uptake intensity by neutrophils, especially for peptide-containing conjugates. This is of tremendous importance for drug delivery in e.g. rheumatoid arthritis. This finding also suggests a positive effect of lipidation on the bio-distribution given that the goal is to keep the therapeutic in the blood stream.
Example 9: Cell Viability Upon Adding the Conjugates
[0309] Cell viability upon adding the conjugates was assessed using Abcam luminescence kit (ab65314 Bioluminescent). The procedure for the assay was performed according to the manufacturer's protocol. Abcam's Cell Viability Assay Kit ab65314 (Bioluminescent) utilizes bioluminescent detection of the ATP levels for a rapid screening of apoptosis and cell proliferation simultaneously in mammalian cells. The assay utilizes luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter. The assay is fully automatic for high throughput (10 seconds/sample). The microtiter plates containing incubation reactions for primary cells with nanoconjugate complexes were analyzed. The initial ATP concentration (before adding conjugate) was 0.15 pM±4%. Cell viability was monitored as low to no change in ATP concentration per well, given in
[0310] The obtained values for conjugates were compared to the data for cells without adding anything and to DAPI data as no toxicity and high toxicity, respectively. Low values of cell viability means high toxicity and vice versa. From the cell viability assay it was found that primary human cells are only little affected by adding conjugates (conjugates vs. DAPI), even in the presence of potentially toxic peptides in the conjugate structure. It was also seen that the lipidation has a positive effect on the viability for conjugates 7, 8 vs 9, 10, at later time point 48 hr.
Example 10: Synthesis of Labelled PAMAM Nanoparticles
[0311] Step 1. Labelling of PAMAM with Sulfo-Cy5.5.
[0312] At the first step, PAMAM G5 was labelled with sulfo-Cy5.5 NHS reagent. Stock solution of the sulfo-Cy5.5 NHS in DMSO (10 mM; 0.5 uL) was added to the solution of the PAMAM/G5 precursor (1 nmol) in 100 mM bicarbonate buffer, pH 8.3 (200 uL). The reaction was kept in dark overnight and purified by dialysis against 20K membrane 2×500 mL mQ, 1 h, and overnight (500 mL MQ).
##STR00008##
[0313] Chemical structure of sulfo-Cyanine 5.5 NHS ester used in step 1.
[0314] Step 2. N-hydroxysuccinimide (15 mM; 100 ul) was incubated with PEG5000 COOH (10 mM; 100 ul), and/or lipid/carbohydrate reagent (10 mM; 100 ul), in MQ water:DMFA 4:1, v/v, over 3 h. The resulting solution was added to 1 mM G5 PAMAM in 1× bicarbonate buffer (pH 8.2; 200 ul), in presence of 15 mM DIC. Both the labelled dendrimer from step 1 and its unlabeled precursors were reacted in separate experiments. The reaction was kept at room temperature under shaking (300 rpm) overnight, and the product was purified by the dialysis against 10K membrane 2×500 mL MQ, and overnight (500 mL MQ).
[0315] Step 3. Amide Coupling with Peptide Antigen
[0316] The coupling was performed as described by Valeur et al., Chem Soc Rev Vol. 38 (2009) pp 606-631. A desired peptide (20 nmol in 300 uL DMSO) was incubated with DCC (30 nmol) and HOBt (30 nmol) for 1 h at room temperature. The resulting mixture was added to the product of step 2 (1 nmol in 200 uL mQ), and the reaction was kept for 2 hr at room temperature, under 250 rpm shaking. The product was purified by the dialysis against 14K membrane using 2×500 mL MQ, and overnight (500 mL MQ).
[0317] The products were analysed by gel electrophoresis, UV-vis absorbance and fluorescence as described below. Concentration of PAMAM in the product was determined by OD255 at pH 8.2. The nanoparticles were characterized by DLS and SEM.
Example 11: Synthesis of Labelled Chitosan-Hyaluronic Acid (CS-HA) Nanoparticles
[0318] In this study the attachment of antigens was done non-covalently owing to high complexation activity of the CS-HA nanoparticle. Lipid and carbohydrate were not added to the complex for this study, since CS and HA are carbohydrates themselves, and because coupling of the lipid was not efficient at the accepted pH range for CS-HA complex.
[0319] Step 1. Encapsulation of DNA/RNA and/or Peptide Antigens
[0320] At pH 6.5, 0.069% w. chitosan (120 kDa) was dissolved in 2 mL 1×PBS, and DNA/RNA (1.2 nmol) and/or peptide (1.2 nmol). The mixture was kept under stirring 1000 rpm for 10 min. The product was purified with Amicon filter device of MWKO 5 kDa following manufacturer's protocol. The product was reconstituted in 2 mL 1×PBS, pH 6.5.
[0321] Step 2. Labelling and PEGylation of Hyaluronic Acid
[0322] Hyaluronic acid (0.15 mg/mL; 10 kDa) was dissolved in 1.7 mL mQ water, and NHS (0.6 μmol, 10 μL of 6.9 mg/mL fresh stock in water) was added. The mixture was stirred overnight at 1000 rpm, and afterwards 1× bicarbonate (pH 8.0; 200 μL), methoxy-PEG-amine (0.3 μmol; Polysciences, 26026-1) and/or sulfo-Cy5.5 amine (0.3 μmol; Lumiprobe) were added, in a total volume of 2.3 mL. The reaction was stirred at room temperature, 1000 rpm, in dark, overnight, and worked up using Amicon 10 kDa MWKO, following the manufacturer's protocol. The product was reconstituted in 2 mL 1×PBS, pH 5. The sulfo-Cy5.5 labelled product should not be heated and may not be exposed to light.
##STR00009##
[0323] Chemical structures of sulfo-Cy5.5 amine and methoxy-PEG5000-amine reagents.
[0324] Step 3. Complexation of Chitosan with Hyaluronic Acid
[0325] The product of step 1 (1 mL) was mixed with the product of step 2 (1 mL) in the buffers mentioned above. The mixture was kept under 1000 rpm shaking, room temperature, for 30 min, and purified by 50 kDa MWKO Amicon, following the manufacturer's protocol.
[0326] The nanoparticles were characterized by DLS and SEM.
Example 12: SLE Mice Study
[0327] The CS-HA-PEG5000-D1 nanoconjugate complex (synthesized as described in example 11) was tested in NZB/W F1 mice: CS-HA-PEG5000-D1 in 1×PBS was administered by IV in the tail vain every 12 h over 2 weeks, at a conjugate dosage of 160 μg/kg animal weight for 10 nM concentration. 30 mice were tested; 80% were female; average age 20 week; average weight/median 20.2 g (18.4 g-23.1 g). Hydroquinone (HQ) was used as control, PO, 2 mg/kg, every 24 h over 2 weeks.
[0328]
[0329] As a control, 20 healthy mice (controls; KO) were treated with the CS-HA-PEG5000-D1 complex; same regimen as described above. Results are presented in
Example 13: Selectivity of Antigens D7 and D8
[0330] The goal was to purify disease associated antibodies using synthetic antigens; and further study selectivity of the antigens.
[0331] Synthetic antigens D7 and D8 (see table 9) were synthesized and their selectivity tested.
TABLE-US-00009 TABLE 9 Synthetic CKD antigens Component Antigen sequence D7 Pre-annealed amino-modified oligosaccharide: NH.sub.2-(ATCG).sub.6:(TAGC).sub.6 (SEQ ID NO. 7) D8 Pre-annealed amino-modified oligosaccharide: NH.sub.2-(TCCT).sub.6:(AGGA).sub.6 (SEQ ID NO. 8)
[0332] SLE antibodies from sera were captured by affinity chromatography using NHS-sepharose and modified antigens as specified in table 9. The protocol of GE Life Science, gravity affinity purification of antibodies, was followed: Column was packed with sepharose, and washed with 0.01% cold HCl; 2 mg/ml ds antigen in 0.1M bicarbonate pH>8 was added; incubated for 1 hour; wash with 10-column volumes NaOAc; wash with 5-10 column volumes 50 mM phosphate buffer pH 7. Sera was pre-treated with CaCl.sub.2)/dextran to remove lipoproteins prior to applying to column. Sera sample was added to column; incubated for 4 min; washed at 0.5 ml/min flow rate with 20 mM PBS, 5 column volumes; and finally SLE antibodies were eluted with 3 column volumes of 100 mM glycine-HCl, 10% dioxane pH 2.5-3.
[0333] Standard ELISA was used to test the selectivity of D7 and D8. ELISA plates comprising antigens D7 and D8, respectively, were tested for their ability to specifically bind the purified SLE-antibodies compared to control samples comprising other antibodies. It was found that especially D8 is selective for SLE antibodies, while D7 was not.
Example 14: Screening of RA Cit-PEP Library
[0334] In autoimmune diseases, epitope-antibody complexes are potent interactions to trigger the specific uptake of a drug. ACPA in particular are intriguing receptors to enter RA associated immune cells. The initial goal was therefore to identify an effective citrullinated peptide epitope for targeting RA associated cells. Table 10 shows the selected twenty-five peptide sequences that have been screened in this work.
TABLE-US-00010 TABLE 10 Citrullinated peptide epitopes Protein PEP # Sequence origin* Comments 1 HHP GIA EFP S(Cit)G KSS SYS KQF fib (SEQ ID No 9) 2 HHP GIA EFP S(Cit)G KSY SYS KQF fib Mutated PEP1 (SEQ ID No 10) 3 HGP GIA EFP S(Cit)G PSY SYS KQF fib Mutated PEP1 (SEQ ID No 11) 4 HGI GLA EFP S(Cit)G KIS AYS KQF fib Mutated PEP1 (SEQ ID No 12) 5 HGP GGA EFP S(Cit)G KAY SYG KQF fib Mutated PEP1 (SEQ ID No 13) 6 AEGGGV(Cit)GPRVVE fib (SEQ ID No 14) 7 ASSGGV(Cit)GPRIVE fib Mutated PEP6 (SEQ ID No 15) 8 AEGASV(Cit)GPRVVE fib Mutated PEP6 (SEQ ID No 16) 9 KDLLPS(Cit)D(Cit)QHLPLIK fib (SEQ ID No 17) 10 KDLLPS(Cit)DGQHLPLIK fib Mutated PEP9 (SEQ ID No 18) 11 KDLLPS(Cit)D(Cit)GAIPLIK fib Mutated PEP9 (SEQ ID No 19) 12 QMRMELE(Cit)PGGNEIT(Cit)GGSTSYG fib (SEQ ID No 20) 13 NVSPGT(Cit)(Cit)EYHTEK fib (SEQ ID No 21) 14 NVAYPT(Cit)(Cit)EYHGEK fib Mutated PEP13 (SEQ ID No 22) 15 ST(Cit)SVSSSSY(Cit)(Cit)MFGG vim (SEQ ID No 23) 16 AAPVSGSSY(Cit)(Cit)MFGG vim Mutated PEP15 (SEQ ID No 24) 17 ST(Cit)SVSSSSYKGAFLG vim Mutated PEP15 (SEQ ID No 25) 18 VYAT(Cit)SSAV(Cit)L(Cit)SSVP vim (SEQ ID No 26) 19 VYATYGSAV(Cit)L(Cit)SSVP vim Mutated PEP18 (SEQ ID No 27) 20 VYAT(Cit)SSAVGLGSSVP vim Mutated PEP18 (SEQ ID No 28) 21 A(Cit)TKQTA(Cit)KSTGGKAP His Citrullinated fragment (SEQ ID No 29) of human histone 3 22 AA(Cit)KSAPSTGGVKKPH His Citrullinated fragment (SEQ ID No 30) of human histone 3 23 Y(Cit)PGTVAL(Cit)EIKKYQKS His Citrullinated fragment (SEQ ID No 31) of human histone 3 24 LI(Cit)KLPFQ(Cit)LV(Cit)EIAQDFK His Citrullinated fragment (SEQ ID No 32) of human histone 3 25 LCAIHAK(Cit)VTIMPKDI His Citrullinated fragment (SEQ ID No 33) of human histone 3 *fib = fibrinogen; vim = vimentin; His = histone.
[0335] The citrullinated peptides epitopes belonged to three major groups, based on the protein they were derived from: fibrinogen (PEP1-PEP14), vimentin (PEP15-PEP20) and histone 3 (PEP21-PEP25) derived peptides. The rationale behind selecting the peptides has been the reported sequences and confirmed activity in RA. Vimentin and fibrinogen are often mutated among individuals. To take this into account, the mutated sequence variants for fibrinogen and vimentin have been recognised using BSI SPIDER homology search software.
[0336] Citrullinated peptide antigens PEP1-PEP25 (SEQ ID No 9-33) (free amine and carboxy-termini) have been purchased from CALSO, Copenhagen, Denmark, and screened in ELISA of a cohort of 30 RA patients, 30 matched healthy controls and 30 patients with systemic lupus erythematosus. The results are shown in
[0337] Next, we compared mutated fibrinogen and vimentin epitopes to native proteins. Prior to ELISA, the mutated epitopes had been confirmed as homologs to the native proteins in NCBI BLAST, with identity score 90-100%. In ELISA, especially mutations in fibrinogen epitopes had a great effect on antibody recognition. On the contrary, mutations in vimentin epitopes had minor to no effect on ACPA binding levels. To the best of our knowledge, this is the first report showing the high influence of mutations within fibrinogen epitopes on ACPA binding. Last, BSI identified no mutants in histone 3 derived sequences, which is in agreement with the fact that histones are highly conservative proteins that rarely mutate.
[0338] Among all tested peptide epitopes, PEP2 with a sequence HHP GIA EFP S(Cit)G KSY SYS KQF (Cit=citrullin) demonstrated a high binding in RA samples (57%), and low to no binding in healthy controls and SLE (0% and 7%). This is in line with previous reports suggesting high relevance of citrullinated fibrinogen to RA.
Example 15: Preparation of PEP2-Nanoconjugates: Chitosan/Hyaluronic Acid/PEG/PEP2
[0339] I) Covalent Attachment of Peptide Via PEG to CS/HA
[0340] Peptide antigen was modified on solid support via C end with COOH-PEG-NH2Fmoc. Fmoc group on PEP2 was deprotected following standard protocol using 20% Piperidine in DMF.
[0341] Covalent conjugation of Hyaluronic acid and PEG-PEP2 product was done via amide bond on PEG. In doing this, 0.5 mg of PEP2-PEG NH2 was coupled with 1 mg Hyaluronic acid at pH 8.3 via NHS/EDC coupling reaction, using 1 mg NHS and 1.3 mg EDC. The mixture was stirred for 6 hrs at 800 rpm. Then, the product was purified with Amicon filter device of Molecular Weight Cut Off (MWCF) 5 kDa following standard protocol for removing residues of NHS/EDC and unconjugated PEG-PEP2. The characterisation of complex was done by MALDI MS and UV VIS. MALDI results showed no peaks of peg peptide followed by absorbance peak of peptide in UV VIS at 280 nm which showed the covalent conjugation of peg peptide with Hyaluronic acid.
[0342] The obtained covalent complex PEP2-PEG-HA was mixed with 3 mg of Chitosan for 1 hr, at 800 rpm. The reaction was quenched with 0.01 M Glycine for 10 min. Samples were then analysed by Nanosight and SEM, given below.
[0343] II) Non-Covalent Attachment of Peptide to CS/HA
[0344] Step 1. Encapsulation of PEP2: At pH 6.5, 0.069% w. chitosan has been dissolved in 2 mL 1×PBS, and PEP2 (1.2 nmol) has been added. The mixture was kept under stirring 1000 rpm for 10 min. The product was purified with Amicon filter device of MWKO 5 kDa following manufacturer's protocol. The product has been reconstituted in 2 mL 1×PBS, pH 6.5.
[0345] Step 2. Labelling and PEGylation of hyaluronic acid: Hyaluronic acid (0.15 mg/mL; 10 kDa) has been dissolved in 1.7 mL mQ water, and NHS (0.6 μmol, 10 μL of 6.9 mg/mL fresh stock in water) had been added. The mixture was stirred overnight at 1000 rpm, and afterwards 1× bicarbonate (pH 8.0; 200 μL), methoxy-PEG-amine (0.3 μmol; Polysciences, 26026-1) were added, in a total volume of 2.3 mL. The reaction was stirred at room temperature, 1000 rpm, in dark, overnight, and worked up using Amicon 10 kDa MWKO, following the manufacturer's protocol. The product has been reconstituted in 2 mL 1×PBS, pH 5.
[0346] Step 3. Complexation of chitosan with hyaluronic acid: Product of step 1 (1 mL) has been mixed with step 2 product (1 mL) in the buffers mentioned above. The mixture was kept under 1000 rpm shaking, room temperature, for 30 min, and purified by 50 kDa MWKO Amicon, following the manufacturer's protocol.
[0347] Nanosight experiment: Nanosight measurement was done in Jang lab, DTU, using Nano sight equipment NTA Version: NTA 3.1 Build 3.1.46 with Script SOP Standard Measurement 03-47-19PM 20D. The cell of the equipment must be cleaned and unscrewed totally by ethanol and Millipore water. 500 μL diluted sample was injected three times for three run and the concentration of Nanoparticles was adjusted using water pH 6 if the concentration of samples doesn't fit the analysis. The size distribution data and the size with maximum number of particles were recorded, see
[0348] Scanning Electron Microscopy (SEM): The morphology of the Chitosan nanoparticles (NP) was investigated using a Quanta FEG 3D scanning electron microscope (SEM). Samples were attached on metal stubs with double-sided adhesive carbon tape and coated with 6 nm of gold for better conductivity using a sputter coater (Leica Coater ACE 200). The average NP diameter was calculated using image J analysis software (National Institutes of Health, MD, USA) measured at different NP for each image.
[0349] The average nanoparticle size was 100-300 nm for the covalently attached complex (
[0350] ELISA Testing of PEP2-Nanoconjugates:
[0351] Prior to ELISA, total amount of protein in each sample was estimated by Bradford method using standard curve of BSA control at known concentration (BioRad). In a maxisorb 96 well plate controls (BSA standard samples at concentrations 2 mg/mL, 1 mg/mL, 0.5 mg/mL and 0.1 mg/mL) and plasma sample were mixed with a Bradford reagent following manufacturer's protocol (BioRad). Plasma samples were used in dilution 1:100. Resulting absorbances at 595 nm were measured on Magellan Tecan microplate reader. Total amount of protein was calculated using standard curve.
[0352] ELISA: Maxisorb 96 well plates (NUNC Thermofisher) were coated with nanoparticle antigens/controls at concentration 8 μg/mL in 1×PBS overnight (room temperature; 100 μl/well). After washing with 1×PT (2×300 μl/well, PT: 50 μl Tween-20 in 1 L 1×PBS), the plates were blocked with 1×PTB (1 h, 37° C.; 100 μl/well, PTB: 20 g BSA, 50 μl Tween-20 in 1 L 1×PBS). Incubation with plasma at desired dilution was performed at room temperature for 1.5 h using diluent: 2 g BSA, 50 μl Tween-20 in 1 L 1×PBS (100 μl/well). This was followed by washing (2×300 μl 1×PBS) and incubation with HPR-labelled secondary antibody for 1.5 h at room temperature using same diluent and dilution of the secondary antibody provided by supplier (HPR-conjugated a-aIgG; Sigma). Subsequent washing (2×300 μl PT) and incubation with freshly prepared TMB-H.sub.2O.sub.2 solution (Sigma; 100 μl/well) was followed by adding a stop solution (1M H2SO4; 50 μl/well) and reading resulting absorbance values at 450 nm on Magellan Tecan microplate reader. Linear range for each antigen was determined via testing series of control dilutions (SLE and healthy controls in dilutions 1:50 to 1:2000). According to the results plasma dilutions 1:100-1:500 were within linear range of the assay for each antigen (R2>0.95).
[0353] Results of ELISA screening for antigen PEP2 and NPs prepared as described above is presented in table 11.
TABLE-US-00011 TABLE 11 Results of ELISA screening for nanoconjugates comprising antigen PEP2 Disease Patients Parameter Healthy control SLE with RA Number of individuals 30 30 30 Female, n (%) 22 (73) 23 (77) 30 (100) Age, median (range) 33.4 (29-56) 33 (20-44) 32 (26-51) Anti-CCP2, 5 (17) 7 (23) 12 (40) n (%).sup.b - commercial ELISA Anti-cit-Fib protein, 2 (7) 4 (13) 15 (50) n (%) - commercial ELISA a-PEP2, n (%) 0 (0) 2 (7) 17 (57) a-NP1, n (%) 1 (3) 2 (7) 17 (57) a-NP2, n (%) 2 (7) 2 (7) 16 (53)