Patent Application
20140273148
1. Field of the Invention
Embodiments of this invention are directed generally to biology, medicine, and immunology. Certain aspects are directed to immunogenic fibrils and their use in inducing an immune response.
2. Description of Related Art
Polypeptides that non-covalently assemble into supramolecular structures, such as nanofibers and nanoparticles, are receiving increased interest as biomaterials for diverse applications, including enzyme catalysis (Wheeldon 2009; Baxa 2002; Patterson 2012; Guglielmi 2009) biosensors (Leng 2010; Men 2009), electronics (Baldwin 2006; Wheeldon 2008), tissue engineering (Wang 2011; Horii 2007), drugs and drug delivery (Webber 2012; Matson 2011; Sinthuvanich 2012), and immunotherapy (Rudra 2010; Hudalla 2013; Black 2012; Wahome 2012). In part, this widespread applicability arises from the ability to incorporate a self-assembling domain and a bioactive ligand, such as a peptide, protein, or nucleic acid, into a single molecule via recombinant genetic fusion or chemical synthesis approaches, without perturbing the assembly or bioactive properties of the respective domains (Cardinale 2012; Lim 2009; Woolfson 2010; Guler 2005). In addition, mixtures of self-assembling polypeptides with or without appended bioactive ligands co-assemble into multi-component biomaterials, in which molecular composition is governed by the molar ratio of polypeptides present during assembly (Collier 2008; Collier 2010; Matson 2012; Minten 2009; Minten 2011). Importantly, this precise and reproducible compositional control enables use of statistical methods to identify ligand formulations that elicit optimal functional responses (Jung 2011), which can be challenging to achieve with co-polymer blends that are subject to compositional drift. However, supramolecular assemblies bearing multiple different folded protein ligands at precise concentrations have not yet been realized, despite existing approaches to create assemblies having tunable concentration of a single protein ligand (Hudalla 2013; Minten 2009; Sangiambut 2013), or bearing two different biologically active proteins (Leng 2010; Men 2009; Minten 2011). A general approach that enables modular and tunable control over integrated protein ligand composition would provide enormous nanofiber design flexibility, ultimately leading to new biomaterials with unique biological or chemical properties for various downstream applications.
Certain embodiments are directed to a nanofiber composition comprising a β-sheet nanofiber structure. The structure may have a length of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200 nm, 0.25, 0.5, 1, 10, 50 to 10, 25, 50, 100 μm, including all values and ranges there between. In certain aspects, the composition has a molecular weight of at least about 1,000, 5,000, 10,000, 100,000 Da to about 1×106, 1×107, 7×108 Da, including all values and ranges there between.
In certain aspects, the β-sheet nanofiber comprises a plurality of a peptide A and a peptide B. The peptide A may be non-β-sheet peptide tags such as a β-sheet fibrillizing tail (a “βTail) exemplified herein. The non-β-sheet peptide may refer to a peptide that forms a structure other than a β-sheet structure when expressed or isolated. The non-β-sheet peptide may be an α-helical peptide or random coil peptide when expressed or isolated. However, the non-β-sheet peptide tags may form a β-sheet structure in the presence of a β-sheet peptide.
In alternative embodiments, the peptide A may be β-sheet peptides (e.g., Q11 peptide) and may be attached to a compound, such as in the form of a fusion protein. A plurality of a peptide A, each is a β-sheet peptide attached to a compound, may form a nanofiber with a plurality of peptide B, which are β-sheet peptides on their own.
In further aspects, the β-sheet nanofiber comprises a peptide B such as a β-sheet peptide. The β-sheet peptide may refer to a peptide that forms a β-sheet structure. The β-sheet peptides may integrate the non-β-sheet peptide tags into a nanofiber structure.
One or more non-β-sheet peptides may be attached as a tag to one or more compounds. It is contemplated that a single tag (such as a single β-tail peptide molecule) may be attached to 1, 2, 3 or more compounds, which may be the same or different with respect to one another. While compositions comprise a plurality of tags, in some embodiments there are same or different tags that are used. Different tags may be attached to the same or to different compounds. Same tags may be attached to the same or to different compounds, for example, each of a subset of the β-tail molecules is attached to a dGFP compound, and each of a subset of the β-tail molecules is attached to a RFP compound, and each of a subset of the β-tail molecules is attached to a dGFP compound. The molar ratio between the non-β-sheet peptide tag and any compound may be 1:1. 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or lower such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or any intermediate ranges. In certain aspects, the composition or structure may be heterogeneous by comprising at least two, three, four, five, six, seven, nine, ten, or more (or any range derivable therein) different compounds. Different compounds may be attached to the same or to different tags. Same compounds may be attached to the same or to different compounds. Moreover, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (or any range derivable therein) different tags.
In some embodiments, the non-β-sheet peptides may comprise one or more α-helical motifs such as coiled-coil motifs. The α-helical motifs may have a sequence of a b c d e f g. The sequence motif may be repeated for about two to seven times. The sequence motifs may be repeated with the same generic structure as described below and same or different specific sequences.
In one, two, three, four, five, six, seven or more or all of the sequence motifs, the position a and d may be non-polar amino acids, hydrophobic amino acids, or non-charged amino acids. For example, a and/or d is Ala (A), Leu (L), Ile (I), Val (V), or a conservative derivative thereof. In particular embodiments, a and/or d is Leu (L) in one, two, three, four, five, six, seven or more or all of the sequence motifs.
In one, two, three, four, five, six, seven or more or all of the sequence motifs, the positions e and g may be charged amino acids, such as Lys (K), Arg (R), His (H), Asp (D), Glu (E) or a conservative derivative thereof. For example, e and g may form or not form attractive electrostatic interactions in one, two, three, four, five, six, seven or more or all of the sequence motifs.
In particular embodiments, one or more of b, c, and, f is a hydrophobic amino acid that favors β-sheet formation or increase β-sheet formation propensity in one, two, three, four, five, six, seven or more or all of the sequence motifs. For example, one or more of b, c, and, f in one or more of the α-helical motifs is Val (V), Tyr (Y), Phe (F), Trp (W), Ile (I), or Thr (T) in one, two, three, four, five, six, seven or more or all of the sequence motifs. More particularly, b, c, and f are beta-sheet forming residues such as Val (V) in one, two, three, four, five, six, seven or more or all of the sequence motifs.
In further aspects, the non-β-sheet peptide comprises an amino acid sequence having at least 50, 60, 70, 75, 80, 85, 90, 95, 99, 100% identity (or any intermediate ranges) with the sequence of LVVLHSELHKLKSEL (SEQ ID NO. 1), LVVLHSHLEKLKSEL (SEQ ID NO. 2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO. 3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO. 4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO. 5). In certain aspects, the non-β-sheet peptide is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 to 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 amino acids in length, including all values and ranges there between.
In certain aspects, one or more of the alpha helical motifs of the non-β-sheet peptide may further comprise at least one, two, three, four, five, six, seven, eight, nine, ten metal binding amino acids or any range derivable therein. Each of the two metal binding amino acids in one or more of the alpha helical motifs or the non-β-sheet peptide may be spaced by at least one, two, three, four, five, six, seven, eight, nine, ten amino acids or any range derivable therein. The spacing amino acids may be any amino acids, hydrophobic, hydrophilic, charged, metal-binding, or not. The non-β-sheet peptide may also not need any metal binding amino acids, such as LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO. 5).
In particular aspects, one or more of the alpha helical motifs of the non-β-sheet peptide further comprise at least two metal binding amino acids spaced by one amino acid. For example, the non-β-sheet peptide may comprise an amino acid sequence having at least or about 50, 60, 70, 75, 80, 85, 90, 95, 99, or 100% identity (or any range derivable therein) with the sequence of LVVLHSHLEKLKSEL (SEQ ID NO. 2) or LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO. 4).
In further aspects, one or more of the alpha helical motifs further comprise at least two metal binding amino acids spaced by three amino acids. For example, the non-β-sheet peptide may comprise an amino acid sequence having at least or about 50, 60, 70, 75, 80, 85, 90, 95, 99, or 100% identity (or any range derivable therein) with the sequence of LVVLHSELHKLKSEL (SEQ ID NO. 1) or LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO. 3).
In further aspects, there may be provided a composition comprising a nanofiber comprising peptide A and peptide B. In further aspects, the peptide A may be attached to any compound at a molar ratio of 1:1. 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or lower such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or any intermediate ranges. The nanofiber may comprise the same compound or two, three, four, five, six or more different compounds.
The peptide A may comprise an amino acid sequence having at least or about 50, 60, 70, 75, 80, 85, 90, 95, 99, or 100% identity (or any range derivable therein) with the sequence of LVVLHSELHKLKSEL (SEQ ID NO. 1), LVVLHSHLEKLKSEL (SEQ ID NO. 2), LKVELEKLKSELVVLHSELHKLKSEL (SEQ ID NO. 3), LKVELEKLKSELVVLHSHLEKLKSEL (SEQ ID NO. 4), or LKVELKELKKELVVLKSELKELKKEL (SEQ ID NO. 5).
The peptide B may comprise an amino acid sequence having at least or about 50, 60, 70, 80, 85, 90, 95, 99, or 100% identity (or any range derivable therein) with the sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33).
For example, the peptide A may be at least, at most or about 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 amino acids in length, including all values and ranges there between. In further aspects, the peptide A may be at least, at most or about 7, 8, 9, 10, 11, 12, 13, 14, 15 to 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 amino acids in length, including all values and ranges there between.
In certain aspects, one or more of the compounds attached to the non-13 sheet peptide tags (or peptide A) may be peptides, polypeptides, nucleic acids, small molecules, antigens, ligands, enzymes, reporters, drugs, matrices, cells, viruses, bacteria, lipids, carbohydrates, or a combination thereof.
For example, one or more of the compounds may be a peptide, same or different. In further aspects, at least one, two, three, four, more or all of the non-β-sheet peptide tags attached to a compound is a fusion protein. The non-β-sheet peptide tag (or peptide A) may be attached to the N and/C terminus of a peptide compound. In particular aspects, the non-β-sheet peptide tag (or peptide A) is attached to a peptide having 2 to 10,000 amino acids in length, more particularly 5, 10, 15, 20 to 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000 amino acids in length, including all values and ranges there between.
Non-limiting examples of a peptide compound attached to a non-β-sheet peptide tag (or peptide A) include an enzyme, fluorescent protein, cell binding domain, cell adhesion domain, extracellular matrix domain, reporter protein, cytokine, antigen, signaling domain, immunomodulating protein, cross-linking protein, hormone, hapten, or a combination thereof. In a particular example, extracellular matrix proteins or extracellular matrix protein domains may be used as the peptide compound or in the composition.
As used herein, the term “extracellular matrix”, abbreviated “ECM”, refers to the complex structural material that is produced by cells in mammalian tissues, particularly cells of connective tissue, for instance such cells as fibroblasts, osteoblasts, chondrocytes, epithelial cells, smooth muscle cells, adipocytes, and mesenchymal cells, and which material in vivo surrounds and supports those cells. Typically, the ECM is composed of fibres embedded in what is commonly referred to as ‘ground substance’. ECM proteins include proteins in the fibers as structural proteins, such as collagen and/or elastin. Particularly suitable collagens are fibril-forming collagens. Type I collagen, type II collagen, type III collagen, type IV collagen or type X collagen are particularly preferred. A particular example is type I collagen.
ECM proteins also include proteins in the ‘ground substance’ of ECM, such as fibrillin, fibronectin, and/or laminin. Additional ECM proteins may incude: glycoproteins such as laminin, entactin, tenascin, fibrillin, or fibronectin, for improving structural integrity of the network and for the attachment of cells to the ECM; osteocalcin (GIa protein), as a protein that binds calcium during mineralization; osteonectin, which serves a bridging function between collagen and mineral component; and sialoproteins, such as bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE).
When used in certain aspects, the term “extracellular matrix” or “extracellular matrix protein or protein domain” refers both to the material in vivo, as well as to the material in isolated form, separated from the cells that produced it. The ECM in certain aspects can be a natural or artificial material (e.g., a proteinaceous or peptide hydrogel).
In particular aspects, the compound attached to one or more of the non-β-sheet peptide tags (or peptide A) is an antigen. Antigens can be microbial antigens, such as viral, fungal, or bacterial; or therapeutic antigens such as antigens associated with cancerous cells or growths, including tumor antigens, or autoimmune disorders. In certain aspects, the antigens are peptides, lipids, carbohydrates, or other immunogenic molecules. The antigens can be T-cell and/or B-cell epitopes.
As used herein, the term “antigen” is a molecule capable of inducing an immune response or of being bound by an antibody or T-cell receptor. An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production of B- and/or T-lymphocytes. The structural aspect of an antigen that gives rise to a biological response is referred to herein as an “antigenic determinant.” B-lymphocytes respond to foreign antigenic determinants via antibody production, whereas T-lymphocytes are the mediator of cellular immunity. Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies, or in the context of an MHC, by T-cell receptors. An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another. In particular aspects, the antigen may be an antigenic determinant or epitope.
In further aspects, the non-β-sheet peptide tags (or peptide A) and β-sheet peptides (or peptide B) may have a molar ratio of about 1:1, 1:2, 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:5000, 1:10,000 50,000 to about 1:100,000 in the composition or the nanofiber structure, including all values and ranges there between.
In certain aspects, β-sheet peptides (or peptide B) comprise a plurality of self-assembling peptides. In other aspects the self-assembling peptides form a beta-sheet rich fibril. In further aspects, the self-assembling peptide comprises an amino acid sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO.12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (Where X=V, A, S, or P) (SEQ ID NO. 25); LLLLKKKKKKKKLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33). In certain aspects, the self-assembling peptide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 to 15, 20, 25, 30, 35, 40, 50, 100, 200, or 500 amino acids in length, including all values and ranges there between. In certain aspects, more than one self-assembling peptide is present in the composition.
In further aspects, the composition or nanostructure may be comprised in a pharmaceutically suitable carrier. For example, the composition may be further defined as an antigen composition. In other aspects, the composition may be in form of a microgel or further defined as microgel.
In further aspects, the composition may not be limited to microgels; particularly the composition may include any 3D structures at a microscopic scale or a macroscopic scale. For example, the composition may be a micro or macro structure with a size, length, diameter of at most, at least, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 nm, μm, mm, cm or any range derivable therein. In certain aspects, the composition may be provided as a 3D cell culture or cell delivery matrix.
There may also be provided methods of providing the compositions described above. For example, the method may comprise mixing non-β-sheet peptide tags (or peptide A) and β-sheet peptides (or peptide B). In particular aspects, a non-β-sheet peptide tag is attached to a compound. In further aspects, same non-β-sheet peptide tag may be attached to same or different compounds as active agents. Therefore, there may be provided a nanofiber complex composition that forms a heterogeneous β-sheet structure comprising the non-β-sheet peptide tags and β-sheet peptides comprising different compounds. In further aspects, the method may further comprise shaking the mixture, thereby forming any form of cell culture or cell delivery matrix such as a microgel or a macrostructure.
The methods may involve a precise control of the concentration, temperature, or pH to achieve a nanostructure with a controlled dosage. For example, the pH may be at least, about, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or any range derivable therein. In particular aspects, the pH may be at about 7. The medium used in the methods may be any aqueous medium, such as phosphate buffered saline. In certain aspects, the peptide B or β-sheet peptides may be used at or above its fibrillizing concentration. For example, at least or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 mM or μM (or any range derivable therein) of the peptide A (or non-β-sheet peptides) or peptide B (or β-sheet peptides) may be used in the methods and compositions described herein.
Certain embodiments are directed to methods of inducing an immune response comprising administering an effective amount of a composition comprising one or more antigens. In further aspects, the method may be provided for treating a patient having or at risk of developing a microbial infection by administering to the patient an effective amount of a composition described herein. In certain aspects, there may be provided methods of treating a patient having or at risk of developing a cancer, comprising administering to the patient an immunotherapy comprising an effective amount of a composition described herein. In further aspects, there may be provided a method of culturing a cell, comprising incubating the cell in a cell culture medium comprising the composition described herein, particularly a macroscopic structure or a microgel composition.
Certain methods and compositions may also be provided for cell delivery, for example, by culturing or suspending cells in the composition provided herein for a period, and then delivering the cells to a tissue or a patient or subject. The composition described herein may be formulated into a cell delivery matrix, for example including ECM proteins or protein domains, particularly from the patient's own body. Methods of delivering cells to a subject include delivering the cell delivery composition to particular tissue sites. For instance, the tissue site may include epithelial, connective, skeletal, muscular, glandular, or nervous tissue. A particular tissue site is cardiac tissue. In an additional aspect of the method, the subject may be a mammal, and in a further aspect the mammal may be a human. One advantage of the cell delivery methods and compositions may be to improve the survival and function of the cells when delivered in vivo. In a particular aspect, at least, at most or about 50, 60, 70, 80, 90, 95, 99% or more (or any range derivable therein) of the cells remain viable after delivery to a tissue site. In a further aspect, the cells are delivered to the tissue site at a constant rate.
Methods may involve administering to the patient or subject at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a pharmaceutical composition or a composition described herein. A dose may be a composition comprising about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (mcg) or μg/ml or micrograms/ml or mM or μM (or any range derivable therein) of each compound or the total amount of a combination of compounds or the compositions.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Certain embodiments are, in part, based on the finding that engineered fusion proteins having a peptide tag such as a β-sheet fibrillizing tail (a “βTail”) integrated into nanofibers of β-sheet fibrillizing polypeptides at a tunable dose, without loss of bioactivity. Further, the inventors discovered that multiple different βTail fusion proteins co-integrated into polypeptide nanofibers, providing biomaterials with modular and precise composition of bioactive proteins. One aspect also involves compositions and methods related to the use of protein-bearing nanofibers as self-adjuvanting vaccines, immunogenic compositions or cell culture medium or structure, with the potential of this approach for creating biomaterials with unique functional properties for various biomedical and biotechnological applications, such as therapeutics, tissue engineering, drug delivery, 3-D culture, biosensors, or electronics.
Embodiments involve the use of non-β-sheet peptide tags. The peptides may form random coil, α-helical structure or β-sheet structure under different conditions. In particular embodiments, the peptide tags may form β-sheet structure in the presence of a β-sheet peptide such as a β-sheet fibrillizing peptide, including self-assembly peptides described below. When isolated or expressed, the peptide tag may form non-β-sheet structures, such as random coil or α-helical structures (e.g., Pagel 2006, incorporated herein by reference).
Such peptide tags may have one or more α-helical motifs or coiled coil motifs, such as heptad repeats. A coiled coil is a structural motif in proteins. The motif may form a plurality of alpha-helices that are coiled together like the strands of a rope (dimers and trimers are the most common types). In particular aspects, the peptides may have intact or modified heptad repeats as described below for forming α-helical structures or favor forming α-helical structures and also β-sheet forming amino acids or amino acids or amino acids that increase the β-sheet formation propensity.
The α-helical motifs may contain a repeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat. The positions in the heptad repeat may be labeled abcdefg, where a and d are the hydrophobic positions, for example being occupied by isoleucine, leucine or valine, b, c, f are likely hydrophilic or polar residues, and e and g are likely charged resides. Using this nomenclature, each heptad may start with any of a, b, c, d, e, f or g (or a′, b′, c′, d′, e′, f′ or g′), not necessarily a or a′. For example, the heptad repeat may be denoted (a-b-c-d-e-f-g)n or (g-a-b-c-d-e-f)n. For example, n may be from about 2, 3, 4, 5, 6, 7, 8, 9 to about 10 or more.
In certain embodiments, positions a and/or d may be leucine (L). In further embodiments, positions e and g may be a pair of charged resides, such as lysine (K) and glutamic acid (E), or glutamic acid (E) and lysine (K), histidine (H) and glutamic acid (E), glutamic acid (E) and histidine (H), arginine (R) and glutamic acid (E), glutamic acid (E) and arginine (R), lysine (K) and aspartic acid (D), aspartic acid (D) and lysine (K), histidine (H) and aspartic acid (D), aspartic acid (D) and histidine (H), arginine (R) and aspartic acid (D) or any other similarly pairs with opposing charges or without opposing charges. In an embodiment, a heptad repeat may have a sequence of (L E K L K S E) ((SEQ ID NO. 34), (L V V L H S E) (SEQ ID NO. 35), or (L H K L K S E) (SEQ ID NO. 36).
Because positions b, c, and f are solvent exposed, these positions only have a minor impact on α-helical or coiled-coil stability. These positions may be any amino acids, such as glutamic acid (E), lysine (K), histidine (H), serine (S), or valine (V). In particular aspects, these positions may comprise one or more β-sheet forming amino acids or amino acids that increase β-sheet formation propensity compared to its absence. Such amino acids may include, but are not limited to glycine, alanine, valine, leucine, and isoleucine, and other non-naturally occurring amino acids which may be used in a similar chemical and structural manner in the peptides.
In further embodiments, the peptide tags may comprise or may not need one or more metal binding sites that may stabilize or disrupt the helical structure or sheet structure. In particular embodiments, the heptad repeats may have one or more metal binding sites introduced in an i, i+4 geometry that stabilizes the helical structure. For example, two metal binding sites, such as histidine (H), may be incorporated into two neighboring heptad repeats. Such metal binding sites may be close enough to each other in the α-helical conformation to help induce or stabilize the α-helical structure and/or slow down the transition to a β-sheet structure.
The peptide tags may comprise from 2 to about 200 (e.g. about 3 to about 100, such as about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 or any intermediate ranges) heptad repeats, or particularly 3, 4, 5, 6, 7, 8, 9 or 10 heptads. Two or more of the heptads may contain the same repeating sequence of seven amino acids. Alternatively, each heptad in the peptide tags may be the same or each may be different.
Folding a sequence with this repeating pattern into an alpha-helical secondary structure may cause the hydrophobic residues to be presented as a “stripe” that coils gently around the helix in left-handed fashion, forming an amphipathic structure. The more favorable way for two such helices to arrange themselves in the water-filled environment of the cytoplasm is to wrap the hydrophobic strands against each other sandwiched between the hydrophilic amino acids. It is thus the burial of hydrophobic surfaces that provides the thermodynamic driving force for the oligomerization. The packing in a coiled-coil interface may be exceptionally tight, with almost complete van der Waals contact between the side chains of the a and d residues.
The α-helices may be parallel or anti-parallel, and may adopt a left-handed super-coil. Although disfavored, a few right-handed α-helices or coiled coils have also been observed in nature and in designed proteins.
Mixing or co-assembly of β-sheet fibrillizing polypeptides that contain a population of peptide tags (particularly referring to a peptide that can form both α-helical structures and β-sheet structures under different conditions) with a different population of β-sheet peptides (a different peptide) may be used to prepare nanofibers, microgels, or scaffolds in certain aspects of the present invention.
Without limitation one or more β-sheet fibrillizing polypeptides comprising different molecules such as antigens may be used to prepare the scaffolds, microgels, and nanofibers. The amount of β-sheet fibrillizing polypeptides comprising non-β-sheet peptides and antigens compared to the β-sheet peptides maybe varied without limitation in the preparation of the scaffolds, microgels, and nanofibers.
The mixture of first and second peptides may be incubated under any conditions suitable for forming nanofibers, microgels, or scaffolds. For example, the condition may be an aqueous medium, a rocking platform, or a combination thereof. The nanofiber may include any forms of nanostructures comprising β-sheet secondary structures, such as a nanofibril, a nanowire, a nanosurface and a nanosphere.
The self-assembled micelles and nanofibers may be characterized by NOE and FT-IR spectroscopy, circular dichroism; nanofiber fiber networks may be visualized using transmission electron microscopy (TEM).
Certain aspects include self-assembling peptides, which may be used in β-sheet peptides (or peptide B) or polypeptides. Non-limiting examples of self-assembling peptides have been described in US Patent Publication 2012-0282292, which is incorporated herein by reference by its entirety.
As used herein, the term “self-assembling peptide” refers to peptides that are able to spontaneously associate and form stable structures. In one embodiment, a self-assembling peptide comprises an amino acid sequence of QQKFQFQFEQQ (SEQ ID NO. 6); QQKFQFQFHQQ (SEQ ID NO. 7); FKFEFKFE (SEQ ID NO. 8); KFQFQFE (SEQ ID NO. 9); QQRFQFQFEQQ (SEQ ID NO. 10); QQRFQWQFEQQ (SEQ ID NO. 11); FEFEFKFKFEFEFKFK (SEQ ID NO. 12); QQRFEWEFEQQ (SEQ ID NO. 13); QQXFXWXFQQQ (Where X denotes ornithine) (SEQ ID NO. 14); FKFEFKFEFKFE (SEQ ID NO. 15); FKFQFKFQFKFQ (SEQ ID NO. 16); AEAKAEAKAEAKAEAK (SEQ ID NO. 17); AEAEAKAKAEAEAKAK (SEQ ID NO. 18); AEAEAEAEAKAKAKAK (SEQ ID NO. 19); RADARADARADARADA (SEQ ID NO. 20); RARADADARARADADA (SEQ ID NO. 21); SGRGYBLGGQGAGAAAAAGGAGQGGYGGLGSQG (SEQ ID NO. 22); EWEXEXEXEX (Where X=V, A, S, or P) (SEQ ID NO. 23); WKXKXKXKXK (Where X=V, A, S, or P) (SEQ ID NO. 24); KWKVKVKVKVKVKVK (SEQ ID NO. 25); LLLL LLLL (SEQ ID NO. 26); VKVKVKVKVDPPTKVKVKVKV (SEQ ID NO. 27); VKVKVKVKVDPPTKVKTKVKV (SEQ ID NO. 28); KVKVKVKVKDPPSVKVKVKVK (SEQ ID NO. 29); VKVKVKVKVDPPSKVKVKVKV (SEQ ID NO. 30); VKVKVKTKVDPPTKVKTKVKV (SEQ ID NO. 31); Fmoc-FF; Fmoc-GG; Fmoc-FG; KKSLSLSLSLSLSLKK (SEQ ID NO. 32); or YTIAALLSPY (SEQ ID NO. 33) or conservatively modified variants thereof. Self-assembling peptides may further comprise other compounds, for example, immunogenic peptides.
Certain peptides that comprise of alternating hydrophilic and hydrophobic amino acids self-assemble to form an exceedingly stable beta-sheet macroscopic scaffold (U.S. Pat. Nos. 5,955,343 and 5,670,483, each of which is incorporated herein by reference).
Many self-complementary peptides have identical compositions and length; such as EAK16, KAE16, RAD16, RAE16, and KAD16 have been exemplified below (Table 1).
The peptides described herein can be chemically synthesized using standard chemical synthesis techniques. In some embodiments the peptides are chemically synthesized by any of a number of fluid or solid phase peptide synthesis techniques known to those of skill in the art. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are well known to those of skill in the art and are described, for example, by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
Certain embodiments comprise incorporating one or more different compounds into a β-sheet assembly via a non-β-sheet peptide that transitions into a β-sheet structure in the presence of β-sheet peptides. The compound can be any biocompatible molecules, like a small molecule, a drug, a peptide, a lipid, a sugar molecule, or a cell, or any bio-compatible material such as a matrix, a gel, membrane, micelle, or fiber. In particular embodiments, the compound may be an antigen. As used herein, the term “biocompatible” refers to a substance which produces no significant untoward effects when applied to, or administered to, a given cell or organism according to the methods and amounts described herein.
A. Compounds
In certain embodiments, the compound may be a peptide. A peptide may be any peptide or polypeptide, including, but not be limited to, an enzyme, fluorescent protein, cell-binding domain, cell adhesion domain, extracellular matrix domain, reporter protein, cytokine, antigen, signaling domain, immunomodulating protein, cross-linking protein, hormone, hapten, or a bioactive ligand, such as a peptide, protein, or nucleic acid.
In further embodiments, the compound includes or is substantially in the form of at least one of an organic or inorganic small molecule, clathrate or caged compound, protocell, coacervate, microsphere, Janus particle, proteinoid, laminate, helical rod, liposome, macroscopic tube, niosome, sphingosome, toroid, vesicular tube, vesicle, small unilamellar vesicle, large unilamellar vesicle, large multilamellar vesicle, multivesicular vesicle, lipid layer, lipid bilayer, micelle, organelle, cell, membrane, nucleic acid, peptide, polypeptide, protein, glycopeptide, glycolipid, lipoprotein, sphingolipid, glycosphingolipid, glycoprotein, peptidoglycan, lipid, carbohydrate, metalloprotein, proteoglycan, chromosome, nucleus, acid, support structure, buffer, protic solvent, aprotic solvent, nitric oxide, nitrous oxide, nitric oxide synthase, amino acid, micelle, polymer, copolymer, monomer, prepolymer, cell receptor, adhesion molecule, cytokine, chemokine, immunoglobulin, antibody, antigen, platelet, extracellular matrix, blood, plasma, cell ligand, zwitterionic material, cationic material, oligonucleotide, nanotube, piloxymer, transfersome, gas, element, contaminant, radioactive particle, hormone, microorganism, bacteria, virus, quantum dot, contrast agent, or any part thereof.
B. Antigens
The term “antigen” may refer to a molecule against which a subject can initiate a humoral and/or cellular immune response. Antigens can be any type of biologic molecule including, for example, simple intermediary metabolites, sugars, lipids, and hormones as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids and proteins.
Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, and other miscellaneous antigens. In certain compositions and methods, the antigen is a peptide.
Viral Antigens.
Examples of viral antigens include, but are not limited to, retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B. and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins E1 and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components; cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpI, gpII, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80% E, and other Japanese encephalitis viral antigen components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fundamental Virology, Second Edition, e's. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.
Bacterial Antigens.
Bacterial antigens which can be used include, but are not limited to, pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diptheria bacterial antigens such as diptheria toxin or toxoid and other diphtheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigen components; hemophilus influenza bacterial antigens such as capsular polysaccharides and other hemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as romps and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.
Fungal Antigens.
Fungal antigens which can be used in the compositions and methods include, but are not limited to, Candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
Parasite Antigens.
Examples of protozoa and other parasitic antigens include, but are not limited to, plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 1 55/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasma antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components.
Tumor Antigens.
Tumor antigens which can be used in the compositions and methods include, but are not limited to, telomerase components; multidrug resistance proteins such as P-glycoprotein; MAGE-1, alpha fetoprotein, carcinoembryonic antigen, mutant p53, immunoglobulins of B-cell derived malignancies, fusion polypeptides expressed from genes that have been juxtaposed by chromosomal translocations, human chorionic gonadotrpin, calcitonin, tyrosinase, papillomavirus antigens, gangliosides or other carbohydrate-containing components of melanoma or other tumor cells. It is contemplated in certain embodiments that antigens from any type of tumor cell can be used in the compositions and methods described herein.
Antigens Relating to Autoimmunity.
Antigens involved in autoimmune diseases, allergy, and graft rejection can be used in certain aspects. For example, an antigen involved in any one or more of the following autoimmune diseases or disorders can be used in the present invention: diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia greata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves opthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis. Examples of antigens involved in autoimmune disease include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor. Examples of antigens involved in allergy include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens such as dust mite antigens and feline antigens, histocompatiblity antigens, and penicillin and other therapeutic drugs. Examples of antigens involved in graft rejection include antigenic components of the graft to be transplanted into the graft recipient such as heart, lung, liver, pancreas, kidney, and neural graft components. An antigen can also be an altered peptide ligand useful in treating an autoimmune disease.
Examples of miscellaneous antigens which can be can be used in certain aspects include endogenous hormones such as luteinizing hormone, follicular stimulating hormone, testosterone, growth hormone, prolactin, and other hormones, drugs of addiction such as cocaine and heroin, and idiotypic fragments of antigen receptors such as Fab-containing portions of an anti-leptin receptor antibody.
C. ECM Proteins or Protein Domains
The compounds may include a cell attachment molecule comprising amino acids, which is an extracellular matrix (ECM) protein, or a peptide that includes an ECM protein domain. As known in the art, ECM proteins provide structural support to cells and/or attach cells that reside in the ECM. Molecules on the surface of cells, such as integrins, carbohydrates, and other cell adhesion molecules can interact with ECM proteins to promote cell attachment. Non-limiting exemplary ECM proteins include fibronectin, laminin, collagen, procollagen, elastin, vitronectin, tenascin, entactin, fibrinogen, thrombospondin, osteopontin (bone sialoprotein), osteocalcin, von Willibrand Factor, and active domains thereof.
ECM protein domains refer to an amino acid sequence found within the ECM protein that, in itself, provides function according to one or more properties of the ECM protein, such as providing structural support to cells and/or for attaching cells. The domain may also be referred to as a “active portion” or “motif.” The peptide that includes an ECM protein domain can have a “core sequence” of amino acid residues, and optionally one or more additional amino acid residues that flank (i.e., on the C-terminus, N-terminus, or both) the core sequence. The one or more additional amino acids that flank the core sequence can correspond to the wild type ECM sequence in the relevant region of the protein, or can be an amino acid(s) that diverges from the wild type sequence (e.g., a “variant amino acid or sequence”). The variant amino acid or sequence can be one that enhances properties of the peptide, such as providing enhanced ligand interaction, and/or can facilitate formation of the second coated layer.
ECM protein domains are known in the art or can be determined using routine experimentation by carrying out assays that are commercially or described in a reference. For example, cell attachment assays which utilize peptides or proteins adhered to plastic or covalently immobilized on a support have been described and can be used to determine the activity of a desired peptide for promoting attachment of cells (see, for example, Malinda, K. M., et al. (1999) FASEB J. 13:53-62; or Kato, R., et al. (2006) J. Biosci. Bioeng. 101:485-95).
Embodiments of the present invention include pharmaceutical compositions comprising the compositions of nanostructures prepared as above and methods for using these compositions. These compositions may be pharmaceutical compositions used in preventive care or therapeutics.
For example, the compositions may be immunogenic compositions used for preventing or ameliorating microbial infections. A particular application is prophylaxis for infectious diseases. Exposure to antigens in the nanostructure can create resistance against such diseases or act as a vaccination for various conditions.
In other embodiments, the compositions may be immunogenic compositions used in immunotherapy, such as a cancer immunotherapy. The antigens used may be antigens that are displayed on tumor cells but not healthy cells. Several antigens have been identified as specific to certain types of tumors, such as Caspase-8, MAGE-I, Tyrosinase, HER-2/neu, and MUC-I. With this in mind, nano structures can be used to deliver such antigens to DCs in lymph nodes as a means for activating T cells to attack tumors. The compositions may be administered to a subject or cells in vivo or cells in vitro. The cells may be immune cells, such as T cells, B cells, NK cells, or any other immune cells.
As such, certain aspects contemplate vaccines and therapeutics for use in active immunization of subjects. Pharmaceutical compositions such as immunogenic compositions can include a β-sheet peptide fibril integrated with non-β-sheet peptide tags coupled to a plurality of antigens, “fibril complex.”
The preparation of pharmaceutical compositions such as vaccine compositions that contain polypeptide or peptide sequence(s) as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. For example, such pharmaceutical may be prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified.
The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the pharmaceutical composition such as vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the compositions. In specific embodiments, vaccines are formulated with a combination of substances, as described in U.S. Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.
Pharmaceutical compositions may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
Upon formulation, compositions may be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and/or immunogenic. The formulations are easily administered in a variety of dosage forms. The quantity to be administered depends on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
The manner of application may be varied widely. Any of the conventional methods for administration of a pharmaceutical composition are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.
In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects of the present invention involve administering an effective amount of a composition to a subject. In some embodiments of the present invention, immunogenic compositions may be administered to the patient to protect against infection by one or more microbial pathogens. Additionally, such compounds can be administered in combination with an antibiotic or other known anti-microbial therapy. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
The compositions and related methods, particularly administration of a peptide fibril/antigen complex may also be used in combination with the administration of traditional therapies. These include, but are not limited to, the administration of antibiotics such as streptomycin, ciprofloxacin, doxycycline, gentamycin, chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin, tetracycline or various combinations of antibiotics. In cancer immunotherapy, the second therapy may be any cancer treatment such as surgery, chemotherapy, gene therapy, or radiation.
In one aspect, it is contemplated that a peptide fibril/immunogen composition and/or therapy is used in conjunction with antibacterial treatment or anticancer therapy. Alternatively, the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agents and/or a proteins is administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and antigenic composition would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for administration significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
Various combinations may be employed, for example antibiotic therapy or cancer therapy is “A” and the immunogenic composition is “B”:
Administration of the immunogenic compositions to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.
The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents, can also be incorporated into the compositions.
The pharmaceutical compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In certain embodiments, a vaccine composition may be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
In addition to the compositions formulated for parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including creams, lotions, mouthwashes, inhalants and the like.
The pharmaceutical compositions can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
Solutions of the pharmaceutical compositions as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in isotonic NaCl solution and either added to hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.
Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
The nanofiber described herein may be used in many known applications employing nanofibers including, but not limited to, filter applications, computer hard drive applications, and pharmaceutical applications as described above. The nanofiber is useful in a variety of biological applications, including cell culture, tissue culture, and tissue engineering and cell delivery applications.
In one application, a nanofibrillar structure for cell culture and tissue engineering may be fabricated using the nanofiber. In an embodiment, the nanofibrillar structure comprises one or more nanofibers, wherein the nanofibrillar structure is defined by a network of one or more nanofibers comprising peptide A and peptide B. In another embodiment, the nanofibrillar structure comprises one or more nanofibers and a substrate wherein the nanofibrillar structure is defined by a network of one or more nanofibers deposited on a surface of the substrate. In another application, any cell culture including a growth media for cell culture may be prepared using the nanofiber.
In an embodiment, the growth media comprises a matrix of nanofibers in the form of a mat, roll, or sheet that may be adapted for insertion into a culture container. In another embodiment, the growth media comprises a matrix of nanofibers that is deposited onto a surface of a culture container or added as a fibrous mesh to the culture container. In another application, the nanofiber may be sprayed or spun onto a three-dimensional structure suitable for cell or tissue culture. The resultant three-dimensional structure is returned to a cell culture apparatus for continued growth where the electrospun fiber structure serves as a platform for growth of the cells.
In a further application, the nanofibers may be electrospun into nonwoven mesh and/or braids for the layered construction of three-dimensional matrices to serve as templates for tissue regeneration. In a further application, the nanofibers may be used as a cell culture medium in high throughput drug analysis and drug sensitivity analysis to increase the number of cells per well providing higher signal for detection of cell response. In another further application, the nanofibers may be used as a cell culture medium in high throughput drug analysis, drug sensitivity analysis, and other therapeutic schemes where the nanofibers provide an environment for the cells to more closely mimic the in vivo nature of the cells in an ex vivo environment.
The nanofibers may be formed as a cell culture or cell delivery matrix, for example, by including ECM proteins or ECM protein domains. In particular aspects, the cell culture or cell delivery matrix may comprise any degradable, bioabsorbable or non-degradable, biocompatible polymer. Exemplary three-dimensional culture or cell delivery materials include polymers and hydrogels comprising collagen, fibrin, chitosan, MATRIGEL™, polyethylene glycol, dextrans including chemically crosslinkable or photocrosslinkable dextrans, and the like. In an embodiment, the three-dimensional culture or cell delivery matrix comprises allogeneic components, autologous components, or both allogeneic components and autologous components. In an embodiment, the three-dimensional culture or cell delivery matrix comprises synthetic or semi-synthetic materials. In an embodiment, the three-dimensional culture or cell delivery matrix comprises a framework or support, such as a fibrin-derived scaffold. The term “scaffold” is used herein to include a wide variety of three-dimensional frameworks, for example, but not limited to a mesh, grid, sponge, foam, or the like.
In certain embodiments, the present invention concerns novel compositions comprising at least one proteinaceous molecule and methods for preparing and using such compositions. The proteinaceous molecules may be used for forming a β-sheet structure or nanofiber or be packaged in the structure as an active compound attached to peptide A or non-β-sheet peptides.
As used herein, a “proteinaceous molecule,” “proteinaceous composition,” “proteinaceous compound,” “proteinaceous chain” or “proteinaceous material” generally refers, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. All the “proteinaceous” terms described above may be used interchangeably herein. For convenience, the terms “protein,” “polypeptide” and “peptide” may be used interchangeably herein.
In certain embodiments the size of the at least one proteinaceous molecule may comprise, but is not limited to, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino molecule residues, and any range derivable therein.
In some aspects the size of a peptide defined in certain aspects of the present invention may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues. In other aspects the size of a peptide may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acid residues, or any range derivable therein. In certain embodiments, peptides less than or equal to 20 amino acids, or peptides 6-10 amino acids in length may be used.
As used herein, an “amino molecule” refers to any amino acid, amino acid derivative or amino acid mimic as would be known to one of ordinary skill in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
Accordingly, the term “proteinaceous composition” encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown on Table 2 below.
In certain embodiments the proteinaceous composition comprises at least one protein, polypeptide or peptide. In further embodiments the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide. As used herein, the term “biocompatible” refers to a substance which produces no significant untoward effects when applied to, or administered to, a given cell or organism according to the methods and amounts described herein. Organisms include, but are not limited to, Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions. In particular embodiments, biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.
Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials.
The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (available at www.ncbi.nlm.nih.gov/). The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
B. Purification or Isolation
In certain embodiments a protein or peptide or a composition comprising such a protein or peptide may be isolated or purified. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue or organ to polypeptide and non-polypeptide fractions. The protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. An example of receptor protein purification by affinity chromatography is disclosed in U.S. Pat. No. 5,206,347, the entire text of which is incorporated herein by reference. A particularly efficient method of purifying peptides is fast performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).
A purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. An isolated or purified protein or peptide, therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.
A peptide, polypeptide or protein that is “purified to homogeneity,” as applied to the present invention, means that the peptide, polypeptide or protein has a level of purity where the peptide, polypeptide or protein is substantially free from other proteins and biological components. For example, a purified peptide, polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully.
Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A particular method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.
To purify a desired protein, polypeptide, or peptide a natural or recombinant composition comprising at least some specific proteins, polypeptides, or peptides may be subjected to fractionation to remove various other components from the composition. Various techniques suitable for use in protein purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like, or by heat denaturation, followed by: centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
Another example is the purification of a specific fusion protein using a specific binding partner. Such purification methods are routine in the art. Certain aspects of the present invention provide DNA sequences for the specific proteins, and any fusion protein purification method may be practiced. However, given many DNA and proteins are known, or may be identified and amplified using the methods described herein, any purification method can now be employed.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type of interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, temperature, etc.). The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
C. Fusion Proteins
Other embodiments of protein conjugates concern fusion proteins. These molecules generally have all or a substantial portion of an antigenic peptide, linked at the N- or C-terminus, to all or a portion of a second polypeptide or protein. For example, fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to, for example, facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
In particular embodiments, the fusion proteins comprise a peptide tag attached to an antigenic protein or peptide. Examples of proteins or peptides that may be incorporated into a fusion protein include cytostatic proteins, cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins and binding proteins. These examples are not meant to be limiting and it is contemplated that within the scope of the present invention virtually any protein or peptide could be incorporated into a fusion protein comprising a peptide tag. Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by chemical attachment using bifunctional cross-linking reagents, by de novo synthesis of the complete fusion protein, or by attachment of a DNA sequence encoding a peptide tag to a DNA sequence encoding the second peptide or protein, followed by expression of the intact fusion protein.
D. Synthetic Peptides
Because of their relatively small size, the peptides in certain aspects can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, 1984; Tam et al., 1983; Merrifield, 1986; Barany and Merrifield, 1979, each incorporated herein by reference. Short peptide sequences, usually from about 6 up to about 35 to 50 amino acids, can be readily synthesized by such methods. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.
E. Linkers/Coupling Agents
If desired, the compound or peptides of interest may be joined via a biologically-releasable bond, such as a selectively-cleavable linker or amino acid sequence. For example, peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated. Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin.
Additionally, while numerous types of disulfide-bond containing linkers are known which can successfully be employed to conjugate moieties, certain linkers will generally be preferred over other linkers, based on differing pharmacologic characteristics and capabilities. For example, linkers that contain a disulfide bond that is sterically “hindered” are to be preferred, due to their greater stability in vivo, thus preventing release of the moiety prior to binding at the site of action.
Additionally, any other linking/coupling agents and/or mechanisms known to those of skill in the art can be used to combine to components or agents of the present, such as, for example, antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.
Cross-linking reagents may be used to form molecular bridges that tie together functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog can be made or that heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
An engineered variant of GFP having a β-sheet fibrillizing tail (i.e. a “βTail”) integrated into nanofibers of β-sheet fibrillizing polypeptides in a βTail-dependent manner without loss of activity (
βTail-GFP integrated into Q11 nanofibers at a precise dose alone, or in combination with other fluorescent βTail fusion proteins, resulting in supramolecular biomaterials with precise composition of multiple different bioactive protein ligands (
In addition, βTail-GFP was not sedimented by centrifugation in the absence of Q11 at any concentration tested, which demonstrated that βTail fusion proteins do not appreciably self-assemble over this concentration range (
Similarly, fusions of βTail-GFPuv, βTail-enhanced GFP (βTail-eGFP), and βTail-dsRED monomer (βTail-dsRED) co-integrated into Q11 nanofibers at predictable concentrations, which correlated with the molar ratio of proteins present in solution during nanofiber assembly (
Together, these results demonstrated that multiple different βTail fusion proteins with a similar tertiary structure co-integrated into synthetic polypeptide nanofibers at a precise dose and in a βTail-dependent manner, without significantly perturbing protein bioactivity.
Polypeptide nanofibers bearing a single protein antigen elicit robust, protein-reactive antibodies in the absence of additional immunostimulatory molecules (Hudalla 2013), and based on the data in this Example, this may be extended to include nanofibers bearing multiple different protein antigens would provide the basis for new subunit vaccines that approach the broad-spectrum, multi-antigen immunity conferred by live or attenuated pathogens
It was demonstrated that a recombinant fusion of βTail and the fungal enzyme cutinase (βTail-cut) integrated into Q11 nanofibers without loss of activity to highlight the versatility of this approach (
Using TEM, the co-assembly of Q11 and βTail into heterogeneous β-sheet nanofibers was characterized in more detail (
Circular dichroism (CD) was used to further characterize Q11 and βTail co-assembly into heterogeneous β-sheets. βTail adopted an α-helical 2° structure in the absence of Q11, and transitioned to a predominantly β-sheet 2° structure in the presence of a 10-fold molar excess of Q11 (
Here, the ability of Q11 nanofibers bearing βTail-GFP to act as self-adjuvanting vaccines provided an initial demonstration of the biomedical potential of these materials (
Taken together, the results demonstrated that engineered fusion proteins having a β-sheet fibrillizing tail integrated into nanofibers of β-sheet fibrillizing polypeptides. Fluorescent and enzymatic proteins, for which bioactivity and tertiary structure are directly linked, were expressed as soluble, bioactive βTail fusion proteins and retained their native bioactivity when integrated into polypeptide nanofibers, which suggested the broad potential of this approach for integrating proteins with diverse biological or chemical activities into supramolecular assemblies. Varying the concentration of βTail fusion proteins present in solution during Q11 self-assembly provided precise control over protein content in the nanofibers, similar to approaches to immobilize folded proteins onto mature nanofibers via affinity tags or covalent capture ligands (Hudalla 2013; Sangiambut 2013). Notably, it was also demonstrated that multiple different βTail fusion proteins co-integrated into polypeptide nanofibers at a precise dose, resulting in supramolecular biomaterials displaying modular and tunable activity related to each protein. Although supramolecular assemblies with precise protein composition have been demonstrated previously (Brodin 2012; King 2012; Sinclair 2011), this could be the first demonstration of integrating multiple different protein ligands at precisely defined doses into supramolecular biomaterials. Importantly, this suggested the enormous potential of this general approach for creating novel biomaterials with unprecedented functional properties, which approach the biomolecular complexity inherent to supramolecular assemblies that govern diverse processes throughout living systems.
Here, it was observed that nanofibers with integrated βTail proteins elicited robust anti-protein antibodies in the absence of additional immunostimulatory molecules, suggesting the potential of these materials for developing new subunit vaccines for disease prophylaxis or immunotherapy. Creating efficacious multi-antigen vaccines is a key objective in modern vaccinology because of the potential for simultaneously raising broad-spectrum immunity against different pathogens or pathogen serotypes, while minimizing the number of immunization administered. An important consideration in the design of multi-antigen vaccines is the potential for “antigenic dominance”, in which adaptive immune responses are selectively elicited against one antigen in a co-administered antigen mixture. The ability to precisely titrate two or more different antigens into polypeptide nanofiber adjuvants is likely to greatly improve efforts to minimize antigenic dominance during optimization of multi-antigen vaccine formulations, when compared to poorly controlled, non-specific antigen adsorption onto conventional adjuvants, such as aluminum hydroxide microparticles. However, in light of the general nature and unprecedented versatility afforded by this approach, it was contemplated that βTail fusion proteins, or analogous strategies, will be widely used to create supramolecular biomaterials for diverse medical and technological applications, including drug delivery, synthetic extracellular matrices for tissue engineering and 3-D culture, biosensors, and beyond.
Peptide Synthesis.
Dimethylformamide, diethyl ether, trifluoroacetic acid (TFA), and dichloromethane were purchased from Fisher Scientific. Piperidine, p-nitrophenyl butyrate, and acetic acid were purchased from Sigma-Aldrich. All amino acids and Rink Amide AM resin were purchased from Novabiochem. The β-sheet fibrillizing polypeptides Q11 (QQKFQFQFEQQ) [SEQ ID NO. 6] (Kolattukudy 1981), HK-Q11 (QQKFQFQFHQQ) [SEQ ID. 7] (
Fusion Protein Construction.
The region encoding cutinase within a pET-21d vector containing the recombinant genetic fusion cutinase-(Gly-Ser linker)-GFPuv-His6 (described previously in Hudalla 2013) was excised from the gene by digestion with NcoI and BamHI restriction enzymes. Complimentary oligonucleotides encoding βTail or βTmutant with an NcoI site at the 5′ end and BamHI at the 3′ end were synthesized by IDT DNA technologies (Iowa), annealed by heating to 95° C. for 3 min then allowing the heating block to cool to room temperature over 60 min, and digested with NcoI and BamHI restriction enzymes. Digested plasmid and oligonucleotides were purified by electrophoresis on a 1% agarose gel, followed by isolation with the QIAquick Gel Extraction kit (Qiagen). Oligonucleotides and plasmid were mixed at a 6:1 molar ratio in T4 DNA ligase buffer (New England Biolabs) heated at 45° C. for 1 min, spiked with 400 units of T4 DNA ligase, and incubated overnight at 18° C. Plasmids were then transformed into OneShot Top10 E. coli (Invitrogen) and grown on LB-agar plates with 100 ug/mL ampicillin. For expression, plasmids were isolated from Top10 E. coli using the QIAquick Miniprep kit (Qiagen), transformed into Origami B (DE3) E. coli, and grown on LB-agar plates with 100 ug/mL ampicillin and 50 ug/mL kanamycin. A pET-21d vector encoding βTail-cut was prepared using a similar method, except a nucleic acid sequence encoding cutinase was amplified out of the vector encoding cutinase-(Gly-Ser linker)-GFP using primers having a 5′ BglII end and a 3′ XhoI end. This gene and the pET-21d vector encoding βTail-(Gly-Ser linker)-GFP were digested with BglII and XhoI, purified, ligated together, and transformed into E. coli. Vectors encoding βTail-eGFP and βTail-dsRED were synthesized and subcloned into pET-21D by Genscript (New Jersey, USA). The sequence of each βTail fusion was confirmed by sequencing performed at the University of Chicago Sequencing Facility (see supplemental materials for fusion protein nucleotide and amino acid sequences).
Fusion Protein Expression and Purification.
Ten milliliters of 2×TY media with 100 μg/mL ampicillin and 50 μg/mL kanamycin A was inoculated with E. coli and maintained overnight at 37° C., 220 rpm. The 10 mL culture was subcultured into 1 L 2×TY with 100 μg/mL ampicillin and 50 μg/mL kanamycin A and maintained at 37° C., 220 rpm until an optical density of 0.6 at λ=600 nm was reached. Protein expression was then induced by adding 0.5 mM IPTG to the culture and maintaining the culture at 37° C., 100 rpm for 4 h. Cells were collected by centrifugation, washed, and lysed into 1×PBS containing 1× BugBuster Protein Extraction Reagent (Novagen, Calif.), 1 eComplete protease inhibitor tablet (Santa Cruz Biotechnology), 300 units DNAse I from bovine pancrease (Sigma-Aldrich, MO), and 100 μg lysozyme for 20 min at room temperature. The lysis buffer was cleared by centrifugation and His 6-tagged βTail fusion proteins were purified from the supernatant using metal-affinity chromatography on His Pur cobalt resin (Thermo Scientific, IL). Protein was eluted from the column with imidazole-containing buffers and concentrated into 1×PBS using centrifugal filter units with a 10,000 DA MWCO (Millipore). Endotoxin content was reduced using Triton X-114 cloud-point precipitation, according to previously reported methods (Hudalla 2013). Briefly, Triton X-114 was added to proteins at a 1:10 (v/v) ratio at 4° C., these solutions were maintained on ice for 20 min, and heated to 37° C. for 10 min. Endotoxin-loaded Triton X-114 micelles were then removed by centrifugation at 5000×g, and the process was repeated two additional times.
Nanofiber Assembly.
Lyophilized Q11 was dissolved in deionized water at a final concentration of 10 mM by vortexing for 5 min. Aqueous Q11 solutions were diluted 10-fold with 1×PBS containing GFP (Vector Labs cat# MB-0752), βTmutant-GFP, or one or more βTail fusion proteins at a total protein concentration between 0.25-1.5 μM, or tryptophan-terminated βTail (W-βTail) at a concentration between 10-100 μM. These solutions were then incubated under static conditions for nanofiber assembly, or on a LabNet Rocker 35 rocker table (speed setting 4) (New Jersey) to induce microgel formation.
Characterizing Protein Integration into Nanofibers.
Q11 nanofibers assembled overnight in the presence of βTail fusion proteins were sedimented by centrifugation at 12000×g for 5 min. The supernatant was removed and analyzed for βTail-GFP, βTmutant-GFP, GFP, or W-βTail content by measuring fluorescence emission with a SpectraMax M5 (excitation 395 nm/emission 503 nm for GFP; excitation 280 nm/emission 325 nm for W-βTail, glass-bottom 96-well plates were used for W-βTail fluorescence measurements), and converting emission intensity to protein concentration using GFP or W-βTail fluorescence standards. Additionally, nanofibers were resuspended in fresh 1×PBS, and nanofiber fluorescence was determined using a SpectraMax M5 plate reader. The μBCA assay kit (Pierce) was used according to the manufacturer's instructions to determine βTail-cut concentration in the supernatant. Protein concentration in the nanofibers was reported as the difference between the protein concentration in solution during nanofiber assembly and the protein concentration in the supernatant after centrifugation.
Fluorescence Microscopy.
Q11 microgel fluorescence was analyzed using a Zeiss Axioscope inverted epifluorescent microscope. Dapi filters were used to visualize βTail-GFPuv; FITC filters were used to visualize βTail-eGFP; and TRITC filters were used to visualize βTail-dsRED. Fluorescence emission of any protein with the inappropriate filter (e.g. βTail-GFPuv with FITC) was negligible (
Cutinase Activity.
Cutinase hydrolyzes the colorless molecule p-nitrophenyl butyrate to the yellow molecule p-nitrophenol, which allows for colorimetric analysis of cutianse activity (Kolattukudy 1981). Cutinase activity of Q11 nanofibers assembled in the presence of 0.25-1.5 μM βTail-cut was analyzed by adding 1 μL of 0.1 M p-nitrophenyl butyrate (Sigma-Aldrich) in dimethyl sulfoxide (Fisher Scientific) to 100 μL of nanofibers in 1×PBS and measuring p-nitrophenol absorbance at 405 nm for 3 min using a SpectraMax M5 plate reader. The initial velocity, vo, of p-nitrophenyl butyrate hydrolysis to p-nitrophenol was calculated from the linear portion of a plot of A405 nm versus time.
Transmission Electron Microscopy.
TEM was performed to visualize βTail-GFP or biotinylated-βTail integration into Q11 nanofibers using previously reported methods (Gasiorowski 2011), with minimal modifications. 1 mM Q11 was assembled in the presence of 1 μM βTail or 1 μM βTail-GFP, or 500 μM Q11 was assembled alone, or in the presence of 50 μM biotinylated-βTail, using methods outlined above. Nanofiber solutions were diluted to 0.25 mM Q11 with 1×PBS, and nanofibers were adsorbed onto 200 mesh lacey carbon grids, blocked with 2% acetylated bovine serum albumin (BSA)/0.1% cold water fish skin gelatin, and placed onto a series of droplets containing (1) monoclonal mouse anti-GFP antibody (Santa Cruz Biotechnology cat. #sc-9996, 1:4 in PBS), (2) goat anti-mouse IgG-15 nm gold particles (EMS cat. #25133), and streptavidin-5 nm gold particles (Invitrogen cat. # A32360, all particles diluted 1:4 in PBS), and (3) 1% uranyl acetate in water. Triplicate PBS washes were performed between staining steps. Grids were analyzed with an FEI Tecnai F30 TEM.
Circular Dichroism.
Circular dichroism was performed using an Aviv303 circular dichroism spectrometer in the University of Chicago Biophysics Core. Solutions containing 25 μM βTail or βTmutant, 250 μM Q11, 25 μM βTail plus 250 μM Q11, or 25 μM βTmutant plus 250 μM Q11 in 1× phosphate buffer plus 120 mM potassium fluoride were analyzed after overnight assembly under static conditions at room temperature. Each sample was analyzed 3 times, and the averaged spectrum was reported.
Immunizations.
Q11 microgels bearing βTail-GFP or βTail-cut were prepared as described above, except the materials were incubated at 4 deg C., rather than room temperature. GFP content in the microgels was analyzed fluorimetrically as described above, and the concentration of GFP in the microgels was used as the basis for βTail-GFP dose in the PBS group. Similarly, cutinase content in the microgels was analyzed with the μABCA assay kit (Pierce), according to the manufacturer's instructions, and βTail-cut dose in the microgels was used as the basis for βTail-cut dose in PBS group. Endotoxin content of all vaccines was analyzed with the Limulus Amoebocyte Lysate assay kit (Lonza) according to the manufacturer's instructions immediately before immunization, and all materials were below the upper limit of 1 EU/mL. Female C57BL/6 mice (6-8 weeks old, Taconic Farms, Ind.) were each given two 50 μL subcutaneous injections near the shoulder blades for each primary and booster immunization, similar to previously reported methods (Hudalla 2013). The GFP dosing regimen was: 90 nmol βTail-GFP with or without 1 mM Q11 at day 0, and 80 nmol βTail-GFP with or without 1 mM Q11 at day 31; the cutianse dosing regimen was: 108 nmol βTail-cut with or without 1 mM Q11 at days 0, 28, and 63. Blood was collected weekly via the submandibular vein. Institutional guidelines for the care and use of laboratory animals were strictly followed under a protocol approved by the University of Chicago's Institutional Animal Care and Use Committee.
ELISA.
ELISA was conducted as previously reported (Hudalla 2013), with minimal modifications. For mice immunized with βTail-GFP, serum collected at week 7 was analyzed; for mice immunized with βTail-GFP, serum collected at week 10 was analyzed. For all total IgG ELISAs, following overnight coating with 1 μg/mL βTmutant-GFP in PBS or PBS (negative control), all plates were washed 3 times with 0.5% Tween-20 in PBS. Wells were then blocked with 200 μL of 1% BSA/0.5% Tween-20 in PBS for 1 h at room temperature. This solution was removed from the wells, 100 μL of serum diluted 1:102-1:109 in PBS with 1% BSA was then added directly to the wells without washing, and the plate was incubated for 1 h at room temperature. At the end of serum binding, the solution was removed from the plate, and the plates were washed 5 times with 0.5% Tween-20 in PBS. 100 μL of peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson Immuno Research, cat 115035003) diluted 1:5000 in PBS with 1% BSA was then added to the wells, and the plates were incubated for 45 min at room temperature. At the end of secondary antibody binding, the solution was removed and plates were washed 5 times with 0.5% Tween-20 in PBS. Plates were then developed by adding 100 μL of TMB substrate (eBioscience CA, cat 00-4201-56), incubating for 7.5 min at room temperature, then quenching the reaction with 50 μL of 1 M H3PO4. Absorbance was then measured at 450 nm with a SpectraMax M5 plate reader (Molecular Devices, CA).
For all isotyping ELISAs, following overnight coating with 1 μg/mL βTmutant-GFP in PBS or PBS, all plates were washed 3 times with 0.5% Tween-20 in PBS. Wells were then blocked with 200 μL of 1% BSA/0.5% Tween-20 in PBS for 1 h at room temperature. This solution was removed from the wells, 100 μL of serum collected at week 7 was diluted 1:500 in PBS with 1% BSA was then added directly to the wells without washing, and the plate was incubated for 1 h at room temperature. At the end of serum binding, the solution was removed from the plate, and the plates were washed 3 times with 0.5% Tween-20 in PBS. 100 μL of goat anti-mouse IgG1 (Sigma cat M5532), IgG2a/c (M5657), IgG2b (M5782), IgG3 (M5907), or IgM (M6157) diluted 1:1000 in PBS with 1% BSA was then added to the wells, and the plates were incubated for 30 min at room temperature. At the end of primary antibody binding, the solution was removed from the plate, and the plates were washed 3 times with 0.5% Tween-20 in PBS. 100 μL of peroxidase-conjugated rabbit anti-goat IgG diluted 1:5000 in PBS with 1% BSA was then added to the wells, and the plates were incubated for 15 min at room temperature. At the end of secondary antibody binding, the solution was removed from the plate, and the plates were washed 3 times with 0.5% Tween-20 in PBS. Plates were then developed by adding 100 μL of TMB substrate, incubating for 5 min at room temperature, then quenching the reaction with 50 μL of 1 M H3PO4. Absorbance was then measured at 450 nm with a SpectraMax M5 plate reader.
All recombinant fusion protein genes were inserted between the NcoI and XhoI sites in pET-21d. Genetic and amino acid sequences for each βTail fusion protein are provided below.
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
The invention was made with government support under Grant Nos. ROl EB009701 and 1R21AI094444 awarded by the National Institutes of Health. The government has certain rights in the invention. This application claims priority to U.S. Provisional Application Ser. No. 61/782,193 filed on Mar. 14, 2013, which is hereby incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61782193 | Mar 2013 | US |
