Patent Application
20190031734
Clostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infections (CDI). CDI is the most common cause for antibiotic-associated diarrhea and the leading cause of gastroenteritis-associated death in developed countries. Existing treatment regimens of CDI with antibiotics are ineffective and the rate of reoccurrence for the disease is high.
Clostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infections (CDI). Utilizing genome-wide CRISPR/Cas9 mediated knockout screen, we identified the Wnt receptors Frizzled (FZD) as TcdB receptors. TcdB competes with Wnt for binding to the conserved cysteine-rich domain (CRD) in FZDs, with the highest affinity toward FZD1, 2, and 7, and is a potent inhibitor of Wnt signaling. A recombinant FZD2-CRD fragment protected cells from TcdB. Triple FZD1/2/7 knockout (KO) cells were dramatically resistant to toxin entry. Thus, FZDs as physiologically relevant epithelial receptors for TcdB and play a role in Wnt signaling blockage in CDI pathogenesis and diseases associated with increased Wnt signaling, e.g., cancer.
One aspect of the present disclosure provides isolated polypeptides comprising an amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 27.
Another aspect of the present disclosure provides isolated polypeptides containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 18.
Another aspect of the present disclosure provides isolated polypeptides containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 19.
Another aspect of the present disclosure provides isolated polypeptides containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 20.
In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.
In some embodiments, the polypeptide is cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycylated, glycosylated, polysialylated, phosphorylated, adenylylated, PEGylated, or combinations thereof. In some embodiments, the polypeptide has a modification at the C-terminus or at the N-terminus.
In some embodiments, the polypeptide further contains a fusion domain. In some embodiments, the fusion domain is selected from the group consisting of polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. In some embodiments, the polypeptide further contains an Fc portion of human IgG1.
Further provided herein are fusion proteins containing: a polypeptide comprising an amino acid sequence that has at least 95%, at least 96, at least 97, at least 98, at least 99, or at least 99.5% identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, which polypeptide is fused to an Fc portion of an immunoglobulin. In some embodiments, the Fc portion is an Fc portion of a human IgG1. In some embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO:21, SEQ ID NO: 22, or SEQ ID NO: 23.
Another aspect of the present disclosure provides chimeric molecules containing a first portion and a second portion, wherein the first portion is an isolated polypeptide disclosed herein, and wherein in the second portion is a molecule that is not the isolated polypeptide disclosed herein.
In some embodiments, the isolated polypeptide binds Frizzled (FZD). In some embodiments, the isolated polypeptide blocks Wnt signaling. In some embodiments, the isolated polypeptide is a dimer, trimer, tetramer, or pentamer. In some embodiments, the isolated polypeptide is attached to a polymer. In some embodiments, the polymer prolongs the serum half-life of the isolated polypeptide. In some embodiments, the polymer prolongs the shelf-life of the isolated polypeptide. In some embodiments, the isolated polypeptide has 1-100 conservative amino acid substitutions.
In some embodiments, the second portion is an anti-bacterial agent. In some embodiments, the anti-bacterial agent is an antibiotic. In some embodiments, the second portion is an antibody that binds Frizzled co-receptors. In some embodiments, the Frizzled co-receptor is lipoprotein receptor-related protein (LRP)-5/6, receptor tyrosine kinase (RTK), or tyrosine-protein kinase transmembrane receptor (ROR2).
In some embodiments, the second portion contains an amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the second portion contains an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
Further provided herein are isolated nucleic acid molecules containing a polynucleotide encoding a polypeptide containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity, or 100% identity of SEQ ID NO: 18.
Further provided herein are nucleic acid molecules comprising a polynucleotide encoding a polypeptide containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity, or 100% identity of SEQ ID NO: 19.
Further provided herein are nucleic acid molecules comprising a polynucleotide encoding a polypeptide containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity, or 100% identity of SEQ ID NO: 20.
Further provided herein are nucleic acid molecules containing a polynucleotide encoding a polypeptide containing an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity, or 100% identity of SEQ ID NO: 21.
Further provided herein are nucleic acid molecules comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity, or 100% identity of SEQ ID NO: 22.
Further provided herein are nucleic acid molecules containing a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity, or 100% identity of SEQ ID NO: 23.
Another aspect of the present disclosure provides pharmaceutical compositions comprising the isolated polypeptides or the chimeric molecules disclosed herein.
In some embodiments, the pharmaceutical composition further contains an additional isolated polypeptide containing an amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the additional isolated polypeptide contains an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the additional isolated polypeptide consists of the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
In some embodiments, the polypeptide is acetylated, carboxylated, glycosylated, phosphorylated, lipidated, acylated, PEGylated, thioglycolic acid amidated, or combinations thereof.
In some embodiments, the polypeptide further comprises a fusion domain. In some embodiments, the fusion domain is selected from the group consisting of polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. In some embodiments, the additional polypeptide comprises an Fc portion of human IgG1. In some embodiments, the fusion domain is an Fc portion of human IgG1.
Another aspect of the present disclosure provides a method of treating Clostridium difficile infection (CDI), the method comprising administering to a subject in need thereof, a therapeutically effective amount of the isolated polypeptide, the chimeric molecule, or the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition further contains an agent that induces Wnt signaling downstream of Frizzled (FZD) in a cell. In some embodiments, the agent is a GSK-3 inhibitor. In some embodiments, the GSK-3 inhibitor is Lithium (LiCl), CHIR99021, SB 216763, BIO, TCS 2002, TC-G 24, TWS 119, SB 415286, A 1070722, AR-A 014418, L803-mts, or combination thereof.
In some embodiments, the pharmaceutical composition further comprises an agent that inhibits the cysteine protease activity of TcdB in a cell. In some embodiments, the agent is ebselen. In some embodiments, the pharmaceutical composition further comprises Frizzled antibodies.
In some embodiments, the cell is a colonic epithelial cell.
Yet another aspect of the present disclosure provides a method of treating cancer, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the isolated polypeptide, the chimeric molecule, or the pharmaceutical composition disclosed herein. In some embodiments, the cancer is colon cancer, lung cancer, liver cancer, or breast cancer.
In some embodiments, the pharmaceutical composition further comprises an agent that blocks Wnt signaling. In some embodiments, the agent is a Dkk family protein, a Secreted Frizzled Related Protein (sFRP), Draxin, IGFBP-4, SOST/Sclerostin, USAG1, or WIF-1. In some embodiments, the agent is an Frizzled antibody. In some embodiments, the cancer is metastatic cancer.
Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Clostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infections (CDI). CDI leads to a range of pathology from diarrhea to life-threatening pseudomembranous colitis and toxic megacolon (1, 2). It is the most common cause for antibiotic-associated diarrhea and the leading cause of gastroenteritis-associated death in developed countries, accounting for nearly a half-million cases and 29,000 deaths annually in the United States (3). Two homologous C. difficile exotoxins, toxin A (TcdA) and toxin B (TcdB), are the causal agents for diseases associated with CDI (4-6). These toxins enter cells via receptor-mediated endocytosis and inactivate small GTPases by glucosylating a key residue, which results in cell-rounding and eventual death of cells (4, 5, 7).
Disclosed herein is the identification of the Wnt receptor Frizzled (FZD) as TcdB receptor. TcdB competes with Wnt for binding to the conserved cysteine-rich domain (CRD) in FZD and functions as a potent inhibitor of Wnt signaling. Binding of TcdB to FZDs directly disrupts the integrity of the colon epithelium and its self-renewal by inhibiting Wnt signaling. In one aspect of the disclosure, we identified regions of TcdB (e.g., TcdB1114-1835) that bind FZD. TcdB1114-1835 is a non-toxic fragment of the TcdB that contains the FZD binding domain but not the enzymatic domains (i.e., the cysteine protease domain or the glucosyltransferase domain), competes with the wild-type TcdB and inhibits wild type TcdB. Thus, the use of TcdB1114-1835 for treating CDI and other diseases is also contemplated.
Without wishing to be bound by any particular mechanism or theory, it is believed that some aspects of the present disclosure relies on, at least in part, a novel mechanisms of Clostridium difficile infection. Such mechanism relates to the role of TcdB in inhibiting Wnt signaling in colonic epithelium cells. Among the two Clostridium difficile toxins, TcdB alone is capable of causing the full spectrum of diseases. However, how TcdB targets the colonic epithelium remains largely undefined due to the lack of established receptors. Chondroitin sulfate proteoglycan 4 (CSPG4, also known as neuron-glial antigen 2 (NG2) in rodents) has been identified as a functional receptor for TcdB in HeLa cells and in a colorectal cell line HT-29. However, CSPG4 is not expressed in colonic epithelial cells. Poliovirus receptor-like 3 (PVRL3) was recently suggested as a cellular factor contributing to necrotic cell death process (cytotoxicity) after exposure to high concentrations of TcdB in HeLa cells and in a colorectal cell line Caco-2, but whether PVRL3 is a relevant TcdB receptor in the colonic epithelium remains unknown and its role in directly mediating TcdB entry into cells has not been established.
Described in the Examples and Figures of the present disclosure are the identification and validation of TcdB receptors in colonic epithelia cells using a CRISPR/Cas9 mediated knockout screening system. The CRISPR/Cas9 system and its use is known in the art, e.g., US Patent Publication US20140357530, the entire contents of which is hereby incorporated by reference. Several Frizzled family proteins (FZDs) are identified and validated as novel and pathologically relevant TcdB receptors in the present disclosure. Among the 10 know FZD proteins, FZD 1, 2, and 7 are identified as the most important TcdB receptors that mediate the pathogenesis of Clostridium difficile. Further, FZD 1, 2, and 7 are redundant receptors for TcdB and have overlapping functions. Binding of TcdB to FZDs mediates the entry of the toxin into the cells. TcdB catalyzes the glycosylation of small GTPases inside epithelial cells, causing cell rounding and death. Accordingly, illustrated herein is a novel mechanism independent of the intracellular mechanism of TcdB pathogenesis, relating to the inhibition of Wnt signaling via competition for the FZD receptors.
FZDs are trans-membrane protein known to be involved in Wnt signaling. These receptors span the plasma membrane seven times and constitute a distinct family of G-protein coupled receptors (GPCRs). FZDs play key roles in governing cell polarity, embryonic development, formation of neural synapses, cell proliferation, and many other processes in developing and adult organisms, many of which relate to the Wnt signaling pathways.
The Wnt signaling pathways are a group of signal transduction pathways comprising proteins that pass signals into a cell through cell surface receptors. Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. All three pathways are activated by binding a Wnt-protein ligand to a Frizzled family receptor, which passes the biological signal to proteins inside the cell. The canonical Wnt pathway leads to regulation of gene transcription. The noncanonical planar cell polarity pathway regulates the cytoskeleton that is responsible for the shape of the cell. The noncanonical Wnt/calcium pathway regulates calcium inside the cell. Wnt signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine).
Wnt signaling was first identified for its role in carcinogenesis, then for its function in embryonic development. Wnt signaling also controls tissue regeneration in adult bone marrow, skin and intestine. For example, Wnt signaling is essential for maintaining colonic stem cells in vivo, which continuously give rise to new epithelial cells. The health of stem cells is critical for maintaining and repairing the epithelium, which turns over at an extraordinary rate: the entire colonic epithelium undergoes complete replacement every 5-7 days. Thus, as illustrated in the present disclosure, during Clostridium difficile infection, inhibition of Wnt signaling pathway led to depletion of colonic stem cells and greatly amplified the damage to the epithelium.
Further provided herein are the regions of FZD that interact with both TcdB and Wnt, resulting in competition. Both TcdB and Wnt bind to an N-terminal extracellular cysteine-rich domain of FZDs (FZD-CRD). TcdB is shown to preferentially bind FZDs 1, 2, and 7. The CRDs of FZDs 1, 2, and 7 are highly conserved with over 98% sequence similarity (See
The region of TcdB that interacts with FZD-CRD is identified to be between amino acid 1501-1830 of the TcdB protein (full-length TcdB protein, SEQ ID NO: 27). Polypeptide fragments corresponding to the region of TcdB that interacts with FZD-CRD, e.g., a polypeptide fragment of TcdB between amino 1114 to 1835 (hereafter termed “TcdB1114-1835”, SEQ ID NO: 18), is able to compete with Wnt and inhibit Wnt signaling, and is lacking the cysteine protease activity and the glucosyltransferase activity of TcdB. Such TcdB1114-1835 polypeptide fragments, prevents the entry of wild-type, pathogenic TcdB from entering the cells. Further, the TcdB1114-1835 fragments that enter the cells, are non-toxic due to its lacking the cysteine protease activity and the glucosyltransferase activity. Additionally, two other non-toxic polypeptides that have similar activity as the TcdB1114-1835 are also provided: TcdB1028-1835 (SEQ ID NO: 19) and TcdB1114-2101 (SEQ ID NO: 20).
In some embodiments, the present disclosure makes available isolated and/or purified forms of polypeptides. “An isolated polypeptide”, as used herein, refers to a polypeptide that is isolated from, or is otherwise substantially free of (e.g., at least 80%, 90%, 95%, 97%, 99%, or 99.5% free of), other protein(s) and/or other polypeptide(s) (e.g., TcdB polypeptide species). In some embodiments, the isolated polypeptides is 100% free of other protein(s) and/or other polypeptide(s) (e.g., TcdB polypeptide species).
The isolated polypeptides of the present disclosure, block or inhibit Wnt signaling in cells. “Block”, or “inhibit”, as used herein, means the amplitude of Wnt signaling is decreased compared to normal physiological condition. Inhibition of Wnt signaling exacerbates the pathological outcome of CDI. Conversely, in certain abnormal or pathological conditions, e.g., cancer, Wnt signaling may also be elevated, or hyperactive compared to normal physiological condition. The amplitude of Wnt signaling under normal physiological condition in different cell types may vary and are known in the art. Abnormal Wnt signaling, or the dysfunction of Wnt signaling pathway, is the underlying mechanism of a variety of diseases. Thus, later in the present disclosure, methods of treating such diseases are contemplated.
In some embodiments, the isolated polypeptides of the present disclosure, comprise an amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 27. In some embodiments, the isolated polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 18. For example, the isolated polypeptide comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 18. In some embodiments, the isolated polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 19. For example, the isolated polypeptide comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 19. In some embodiments, the isolated polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 20. For example, the isolated polypeptide comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 20. In some embodiments, the isolated polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 18. In some embodiments, the isolated polypeptide comprises an amino acid sequence that has 85%, 86%, 87&, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 19. In some embodiments, the isolated polypeptide comprises an amino acid sequence that has 85%, 86%, 87&, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 20. In some embodiments, the isolated polypeptide consists of an amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.
The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NB LAST) can be used.
The polypeptides described herein can be conjugated or otherwise covalently attached to other molecules (e.g., using a chemical linker). One such form of attachment is through a non-amide linkage (e.g., a disulfide bond). In some embodiments, the polypeptide is covalently attached (e.g., via a linker molecule) to an antibody or a domain thereof suitable for enhancing the half-life of the molecule (e.g., one or more constant domains in an Fc domain). In some embodiments, the polypeptide is linked to an Fc domain disclosed herein (e.g., IgG, IgA, IgM, IgD, or IgE).
In some embodiments, the isolated polypeptide of the present disclosure, further comprises a fusion domain. Thus, also provided herein are functional variants or modified forms of the polypeptide fragments having one or more fusion domains. Well known examples of such fusion domains include, without limitation, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QlAexpress™ system (Qiagen) useful with (HIS6) fusion partners. In some embodiments, the isolated polypeptide fragment is fused with a domain that stabilizes the isolated polypeptide fragment in vivo (a “stabilizer” domain). “Stabilizing”, as used herein, means an increase in the half-life of the polypeptide in vivo, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains.
In some embodiments, the isolated polypeptides of the present disclosure, further comprises an Fc portion of human IgG1 (SEQ ID NO: 28). Thus, fusion proteins an Fc portion of an immunoglobulin are also contemplated herein. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence that has at least 95% identity to SEQ ID NO: 18, wherein the said polypeptide is fused to an Fc portion of an immunoglobulin. For example, the polypeptide in the fusion protein of the present disclosure, may comprise an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 18. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence that has 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence that has at least 95% identity to SEQ ID NO: 19, wherein the said polypeptide is fused to an Fc portion of an immunoglobulin. For example, the polypeptide in the fusion protein of the present disclosure, may comprise an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 19. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence that has 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 19. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence that has at least 95% identity to SEQ ID NO: 20, wherein the said polypeptide is fused to an Fc portion of an immunoglobulin. For example, the polypeptide in the fusion protein of the present disclosure, may comprise an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 20. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence that has 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 20. In some embodiments, the fusion protein comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, fused to the Fc portion of a human IgG1. Also provided herein are exemplary fusion proteins comprising a TcdB1114-1835 polypeptide fused to an Fc domain (SEQ ID NO: 21), a TcdB1028-1835 polypeptide fused to an Fc domain (SEQ ID NO: 22), and a TcdB1114-2101 polypeptide fused to an Fc domain (SEQ ID NO: 23). The exemplary isolated polypeptide fragment is provided for the sole purpose of illustration and is not meant to be limiting.
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Fc domain is
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEK
TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK (Fc domain is underlined)
Optionally, the Fc domain may have one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domain having one or more of these mutations (e.g., Asp-265 mutation) has reduced ability of binding to the Fc receptor relative to a wildtype Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., Asn-434 mutation) has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fc domain.
It is understood that different elements of the fusion proteins may be arranged in any manner that is consistent with the desired functionality. For example, the TcdB1114-1835 polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to a TcdB1114-1835 polypeptide. The TcdB1114-1835 polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
As used herein, the term, “immunoglobulin Fc region” or simply “Fc” is understood to mean the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment the immunoglobulin Fc region comprises at least an immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably lacks the CH1 domain.
In some embodiments, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). Other classes of immunoglobulin, IgA (Iga), IgD (Igδ), IgE (Igε) and IgM (Igμ), may be used. The choice of appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CH3 domain of Fc γ or the homologous domains in any of IgA, IgD, IgE, or IgM.
Furthermore, it is contemplated that substitution or deletion of amino acids within the immunoglobulin heavy chain constant regions may be useful in the practice of the methods and compositions disclosed herein. One example would be to introduce amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for Fc receptors (Cole et al. (1997) J. Immunol. 159:3613).
Optionally, the isolated polypeptides of the present disclosure, may comprise modifications. Polypeptides comprising modifications have additional features other than amino acid contents. As used herein, a “modification” or “derivative” of a peptide produces a modified or derivatized polypeptide, which is a form of a given peptide that is chemically modified relative to the reference peptide, the modification including, but not limited to, oligomerization or polymerization, modifications of amino acid residues or peptide backbone, cross-linking, cyclization, conjugation, pegylation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolic acid amidation, alkylation, methylation, polyglycylation, glycosylation, polysialylation, adenylylation, PEGylation, fusion to additional heterologous amino acid sequences, or other modifications that substantially alter the stability, solubility, or other properties of the peptide while substantially retaining the activity of the polypeptides described herein. It is to be understood that the isolated polypeptides comprising such modifications, are cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycylated, glycosylated, polysialylated, phosphorylated, adenylylated, PEGylated, or combination thereof. As a result, the modified polypeptide fragments of the present disclosure may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. The isolated polypeptides of the present disclosure, may comprise the modifications disclosed herein at the C-terminus (e.g., C-terminal amidation), N-terminus (e.g., N-terminal acetylation). Terminal modifications are useful, and are well known, to reduce susceptibility to proteinase digestion, and therefore serve to prolong half-life of the polypeptides in solutions, particularly biological fluids where proteases may be present. In some embodiments, the polypeptides or fusion proteins described herein are further modified within the sequence, such as, modification by terminal-NH2 acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications.
Terminal modifications are useful, to reduce susceptibility by proteinase digestion, and therefore can serve to prolong half-life of the polypeptides in solution, particularly in biological fluids where proteases may be present. Amino terminus modifications include methylation (e.g., —NHCH3 or —N(CH3)2), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO— or sulfonyl functionality defined by R—SO2-, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.
Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the peptides described herein, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. Methods of circular peptide synthesis are known in the art, for example, in U.S. Patent Application No. 20090035814; Muralidharan and Muir, 2006, Nat Methods, 3:429-38; and Lockless and Muir, 2009, Proc Natl Acad Sci USA. June 18, Epub. C-terminal functional groups of the peptides described herein include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
In some embodiments, the polypeptides or the fusion proteins described herein are phosphorylated. One can also readily modify peptides by phosphorylation, and other methods (e.g., as described in Hruby, et al. (1990) Biochem J. 268:249-262). One can also replace the naturally occurring side chains of the genetically encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for instance with groups such as alkyl, lower (C1-6) alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocycles. In particular, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
In some embodiments, the isolated polypeptide of the present disclosure is multimeric, e.g., a dimer, trimer, tetramer, or pentamer. In some embodiments, the molecular linker used for forming the oligomeric polypeptides is a peptide linker molecule. In some embodiments, the peptide linking molecule comprises at least one amino acid residue which links at least two peptides according to the disclosure. The peptide linker comprises, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids residues and preferably less than 50 amino acids residues. The peptide linking molecule can couple polypeptides or proteins covalently or non-covalently. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. A peptide linker is attached on its amino-terminal end to one peptide, polypeptide or polypeptide domain (e.g., a C-peptide) and on its carboxyl-terminal end to another peptide, polypeptide or polypeptide domain (again, e.g., a C-peptide). Examples of useful linker peptides include, but are not limited to, glycine polymers ((G)n) including glycine-serine and glycine-alanine polymers (e.g., a (Gly4Ser)n repeat where n=1-8, preferably, n=3, 4, 5, or 6). Other examples of peptide linker molecules are described in U.S. Pat. No. 5,856,456 and are hereby incorporated by reference.
In another embodiment, the molecular linker is a chemical linker such as linkages by disulfide bonds between cysteine amino acid residues or by chemical bridges formed by amine crosslinkers, for example, glutaraldehyde, bis(imido ester), bis(succinimidyl esters), diisocyanates and diacid chlorides. Extensive data on chemical cross-linking agents can be found at INVITROGEN's Molecular Probe under section 5.2.
In certain embodiments, the peptide monomers described herein are dimerized or multimerized by covalent attachment to at least one linker moiety. The linker moiety is preferably, although not necessarily, a C1-12 linking moiety optionally terminated with one or two —NH— linkages and optionally substituted at one or more available carbon atoms with a lower alkyl substituent. Preferably the linker comprises —NH—R—NH— wherein R is a lower (C1-6) alkylene substituted with a functional group, such as a carboxyl group or an amino group, that enables binding to another molecular moiety (e.g., as may be present on the surface of a solid support during peptide synthesis or to a pharmacokinetic-modifying agent such as PEG). In certain embodiments the linker is a lysine residue. In certain other embodiments, the linker bridges the C-termini of two peptide monomers, by simultaneous attachment to the C-terminal amino acid of each monomer. In other embodiments, the linker bridges the peptides by attaching to the side chains of amino acids not at the C-termini. When the linker attaches to a side chain of an amino acid not at the C-termini of the peptides, the side chain preferably contains an amine, such as those found in lysine, and the linker contains two or more carboxy groups capable of forming an amide bond with the peptides.
The polypeptides, fusion proteins, and polypeptide multimers as described herein may be attached to one or more polymer moieties. Preferably, these polymers are covalently attached to the polypeptides of the disclosure. Preferably, for therapeutic use of the end product preparation, the polymer is pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer-peptide conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.
Suitable polymers include, for example, polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Such a polymer may or may not have its own biological activity. The polymers can be covalently or non-covalently conjugated to the polypeptide. Methods of conjugation for increasing serum half-life and for radiotherapy are known in the art, for example, in U.S. Pat. Nos. 5,180,816, 6,423,685, 6,884,780, and 7,022,673, which are hereby incorporated by reference in their entirety.
In some embodiments, the polypeptides monomers, dimers, or multimers as described herein may be attached to one or more water soluble polymer moieties. The water soluble polymer may be, for example, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), poly(n-vinyl-pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, and polyoxyethylated polyols. A preferred water soluble polymer is PEG.
The polymer may be of any molecular weight, and may be branched or unbranched. The average molecular weight of the reactant PEG is preferably between about 3,000 and about 50,000 daltons (the term “about” indicating that in preparations of PEG, some molecules will weigh more, and some less, than the stated molecular weight). More preferably, the PEG has a molecular weight of from about 10 kDa to about 40 kDa, and even more preferably, the PEG has a molecular weight from 15 to 30 kDa. Other sizes may be used, depending on the desired therapeutic profile (e.g., duration of sustained release desired; effects, if any, on biological activity; ease in handling; degree or lack of antigenicity; and other effects of PEG on a therapeutic peptide known to one skilled in the art).
The number of polymer molecules attached may vary; for example, one, two, three, or more water-soluble polymers may be attached to a peptide of the disclosure. The multiple attached polymers may be the same or different chemical moieties (e.g., PEGs of different molecular weight).
In certain embodiments, PEG may be attached to at least one terminus (N-terminus or C-terminus) of a peptide monomer or dimer. In other embodiments, PEG may be attached to a linker moiety of a peptide monomer or dimer. In a preferred embodiment, PEG is attached to the linker moiety of a peptide dimer. Optionally, the linker contains more than one reactive amine capable of being derivatized with a suitably activated PEG species.
In some embodiments, the isolated polypeptides, fusion proteins, or polypeptide multimers described herein, whether monomeric, oligomeric or cyclic, is PEGylated. PEGylation is the process of covalent attachment of Polyethylene glycol polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can “mask” the agent from the host's immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins. PEGylation, by increasing the molecular weight of a molecule, can impart several significant pharmacological advantages over the unmodified form, such as: improved drug solubility, reduced dosage frequency, without diminished efficacy with potentially reduced toxicity, extended circulating life, increased drug stability, and enhanced protection from proteolytic degradation. In addition, PEGylated drugs are have wider opportunities for new delivery formats and dosing regimens. Methods of PEGylating molecules, proteins and peptides are well known in the art, e.g., as described in U.S. Pat. Nos. 5,766,897; 7,610,156; 7,256,258 and the International Application No. WO/1998/032466.
Encompassed herein are conjugates of the polypeptide described herein or of a variant or derivative thereof. These polypeptides can be conjugated to other polymers in addition to polyethylene glycol (PEG). The polymer may or may not have its own biological activity. Further examples of polymer conjugation include but are not limited to polymers such as polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Conjugation to a polymer can improve serum half-life, among other effects. A variety of chelating agents can be used to conjugate the peptides described herein. These chelating agents include but are not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA), ethyleneglycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA), N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid (TITRA), 1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA), and 1,4,8,11-tetraazacyclotetradecane (TETRA). Methods of conjugation are well known in the art, for example, P. E. Thorpe, et. al, 1978, Nature 271, 752-755; Harokopakis E., et. al., 1995, Journal of Immunological Methods, 185:31-42; S. F. Atkinson, et. al., 2001, J. Biol. Chem., 276:27930-27935; and U.S. Pat. Nos. 5,601,825, 5,180,816, 6,423,685, 6,706,252, 6,884,780, and 7,022,673, which are hereby incorporated by reference in their entirety.
In some embodiments, the polymer prolongs the serum half-life of the isolated polypeptide when attached to the isolated polypeptide. In some embodiments, the polymer prolongs the shelf-life of the isolated polypeptide when attached to the isolated polypeptide. The “serum half-life” of an isolated polypeptide, as used herein, refers to the period of time required for the concentration or amount of the polypeptides in the body to be reduced by one-half. A polypeptide's serum half-life depends on how quickly it is eliminated from the serum. The longer the serum half-life is, the more stable the polypeptide is in the body. The “shelf-life”, refers to the period of time, from the date of manufacture, that a product is expected to remain within its approved product specification while stored under defined conditions. It is desirable for a therapeutic agent, e.g., the isolated polypeptide of the present disclosure, to have a longer shelf-life.
Other methods for stabilizing peptides known in the art may be used with the methods and compositions described herein. For example, using D-amino acids, using reduced amide bonds for the peptide backbone, and using non-peptide bonds to link the side chains, including, but not limited to, pyrrolinone and sugar mimetics can each provide stabilization. The design and synthesis of sugar scaffold peptide mimetics are described by Hirschmann et al. (J. Med. Chem., 1996, 36, 2441-2448, which is incorporated herein by reference in its entirety). Further, pyrrolinone-based peptide mimetics present the peptide pharmacophore on a stable background that has improved bioavailability characteristics (see, for example, Smith et al., J. Am. Chem. Soc. 2000, 122, 11037-11038), which is incorporated herein by reference in its entirety.
The isolated polypeptides of the present disclosure, may comprise conservative amino acid substitutions. A “conservative amino acid substitution”, refers to an amino acid substitution that changes an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size). Conservative substitutions of amino acids include, for example, substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Conservative amino acid substitutions do not alter the relative charge or size characteristics of the protein in which the amino acid substitutions are made. Conservative amino acid substitutions typically do not change the overall structure of the peptide and/or the type of amino acid side chains available for forming van der Waals bonds with a binding partner. In some embodiments, the isolated polypeptide may comprise 1-100 conservative amino acid substitutions. For example, the isolated polypeptide may comprise 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, or 100 conservative amino acid substitutions.
Amino acid substitution can be achieved during chemical synthesis of the peptide by adding the desired substitute amino acid at the appropriate sequence in the synthesis process. Alternatively, molecular biology methods can be used. Non-conservative substitutions are also encompassed to the extent that they substantially retain the activities of those peptides described herein.
The amino acid substituted polypeptide will substantially retain the activity of the non-substituted polypeptide. By “substantially retain” means one or more activity of the variant is at least 50% compared to the activity of the original polypeptide in a similar assay, under similar conditions; preferably the activity is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold or higher activity compared to the original polypeptide.
All combinations of the different modifications and derivativizations are envisioned for the polypeptides, fusion proteins and oligomer polypeptides described herein. Modifications, derivatives and methods of derivatizing polypeptides are described in Published International Application WO 2010/014616, the contents of which are incorporated herein by reference.
Other aspects of the present disclosure provide chimeric molecules comprising a first portion and a second portion, wherein the first portion is any isolated polypeptides, fusion proteins, multimeric polypeptides, or variants/derivatives disclosed herein. It is to be understood that the second portion of the chimeric molecule is not the same polypeptide as the first portion of the chimeric molecule. In some embodiments, the first portion of the chimeric molecule is an isolated polypeptide binds Frizzled (FZD). In some embodiments, binding of the isolated polypeptides to FZDs blocks Wnt signaling pathways.
In some embodiments, the second portion of the chimeric molecule comprises a therapeutic agent. In some embodiments, the therapeutic agent may be an anti-bacterial agent. In some embodiments, the therapeutic agent may be an antibiotic. Classes of anti-bacterial agents that may be used in accordance with the present disclosure include, without limitation, aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, quinolones, sulfonamides, and tetracyclines. It is to be understood that any known anti-bacterial agent in the art that can be attached to a polypeptide may be used herein.
In some embodiments, the second portion of the chimeric molecule may be a binder or antibody that binds the Frizzled co-receptors. It is known in the art that to facilitate Wnt signaling, co-receptors may be required alongside the interaction between the Wnt protein and FZDs. Upon activation of the receptor, a signal is sent to the phosphoprotein Dishevelled (Dsh), which is located in the cytoplasm. Blocking of the Frizzled co-receptors via binding of an antibody also blocks Wnt signaling. Examples of Frizzled co-receptors include, without limitation, lipoprotein receptor-related protein (LRP)-5/6, receptor tyrosine kinase (RTK), and tyrosine-protein kinase transmembrane receptor (ROR2). Thus, antibodies to the Frizzled co-receptors described herein, may be used as the second portion of the chimeric molecule of the present disclosure, the facilitate the blocking of Wnt signaling at the receptor level.
In some embodiments, the second portion of the chimeric molecule may be a FZD-CRD fused to the polypeptide of the first portion. In some embodiments, the second portion comprises an amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the second portion of the chimeric molecule comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 24. In some embodiments, the second portion of the chimeric molecule comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 24. In some embodiments, the second portion of the chimeric molecule comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 25. In some embodiments, the second portion of the chimeric molecule comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 25. SEQ ID NO: 25. In some embodiments, the second portion of the chimeric molecule comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 26. In some embodiments, the second portion of the chimeric molecule comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 26.
The isolated polypeptides of the present disclosure (e.g., polypeptides comprising amino acid sequence of any of SEQ ID NOs: 18-26), will generally be produced by expression form recombinant nucleic acids in appropriate cells (e.g., E. coli, or insect cells) and isolated. The nucleic acids encoding the polypeptides described herein may be obtained, and the nucleotide sequence of the nucleic acids determined, by any method known in the art. Further provided herein are isolated and/or recombinant nucleic acids encoding any of the isolated polypeptide fragments disclosed herein. For example, SEQ ID NO: 29 encodes the TcdB1114-1835 polypeptide. The nucleic acids encoding the isolated polypeptide fragments of the present disclosure, may be DNA or RNA, double-stranded or single stranded.
In certain aspects, the subject nucleic acids encoding the isolated polypeptide fragments are further understood to include nucleic acids encoding polypeptides that are variants of SEQ ID NOs: 18 to 23. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity of SEQ ID NO: 18. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity of SEQ ID NO: 19. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity of SEQ ID NO: 20. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity of SEQ ID NO: 21. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity of SEQ ID NO: 22. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity of SEQ ID NO: 23. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of SEQ ID NO: 18. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of SEQ ID NO: 19. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of SEQ ID NO: 20. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of SEQ ID NO: 21. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of SEQ ID NO: 22. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of SEQ ID NO: 23.
In some embodiments, the nucleic acid is comprised within a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid.
A variety of promoters can be used for expression of the polypeptides described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.
Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.
Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used (Yao et al., Human Gene Therapy; Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).
Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.
An expression vector comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the polypeptides described herein. In some embodiments, the expression of the polypeptides described herein is regulated by a constitutive, an inducible or a tissue-specific promoter.
The host cells used to express the isolated polypeptides described herein may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells. In particular, mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al. (1986) “Powerful And Versatile Enhancer-Promoter Unit For Mammalian Expression Vectors,” Gene 45:101-106; Cockett et al. (1990) “High Level Expression Of Tissue Inhibitor Of Metalloproteinases In Chinese Hamster Ovary Cells Using Glutamine Synthetase Gene Amplification,” Biotechnology 8:662-667).
A variety of host-expression vector systems may be utilized to express the isolated polypeptides described herein. Such host-expression systems represent vehicles by which the coding sequences of the isolate d polypeptides described herein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the isolated polypeptides described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for the isolated polypeptides described herein; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the isolated polypeptides described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) containing the sequences encoding the isolated polypeptides described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the isolated polypeptides described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the polypeptides being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of polypeptides described herein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Rüther et al. (1983) “Easy Identification Of cDNA Clones,” EMBO J. 2:1791-1794), in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye et al. (1985) “Up-Promoter Mutations In The lpp Gene Of Escherichia Coli,” Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989) “Expression Of Human Asparagine Synthetase In Escherichia Coli,” J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (e.g., see Logan et al. (1984) “Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987) “Expression And Secretion Vectors For Yeast,” Methods in Enzymol. 153:516-544).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. For example, in certain embodiments, the polypeptides described herein may be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor), requiring proteolytic cleavage by native or recombinant cellular mechanisms to form separate polypeptides described herein. The disclosure thus encompasses engineering a nucleic acid sequence to encode a polyprotein precursor molecule comprising the polypeptides described herein, which includes coding sequences capable of directing post translational cleavage of said polyprotein precursor. Post-translational cleavage of the polyprotein precursor results in the polypeptides described herein. The post translational cleavage of the precursor molecule comprising the polypeptides described herein may occur in vivo (i.e., within the host cell by native or recombinant cell systems/mechanisms, e.g. furin cleavage at an appropriate site) or may occur in vitro (e.g. incubation of said polypeptide chain in a composition comprising proteases or peptidases of known activity and/or in a composition comprising conditions or reagents known to foster the desired proteolytic action). Purification and modification of recombinant proteins is well known in the art such that the design of the polyprotein precursor could include a number of embodiments readily appreciated by a skilled worker. Any known proteases or peptidases known in the art can be used for the described modification of the precursor molecule, e.g., thrombin or factor Xa (Nagai et al. (1985) “Oxygen Binding Properties Of Human Mutant Hemoglobins Synthesized In Escherichia Coli,” Proc. Nat. Acad. Sci. USA 82:7252-7255, and reviewed in Jenny et al. (2003) “A Critical Review Of The Methods For Cleavage Of Fusion Proteins With Thrombin And Factor Xa,” Protein Expr. Purif. 31:1-11, each of which is incorporated by reference herein in its entirety)), enterokinase (Collins-Racie et al. (1995) “Production Of Recombinant Bovine Enterokinase Catalytic Subunit In Escherichia Coli Using The Novel Secretory Fusion Partner DsbA,” Biotechnology 13:982-987 hereby incorporated by reference herein in its entirety)), furin, and AcTEV (Parks et al. (1994) “Release Of Proteins And Peptides From Fusion Proteins Using A Recombinant Plant Virus Proteinase,” Anal. Biochem. 216:413-417 hereby incorporated by reference herein in its entirety)) and the Foot and Mouth Disease Virus Protease C3.
Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express polypeptides described herein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the polypeptides described herein. Such engineered cell lines may be particularly useful in screening and evaluation of polypeptides that interact directly or indirectly with the polypeptides described herein.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) “Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells,” Cell 11: 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1992) “Use Of The HPRT Gene And The HAT Selection Technique In DNA-Mediated Transformation Of Mammalian Cells First Steps Toward Developing Hybridoma Techniques And Gene Therapy,” Bioessays 14: 495-500), and adenine phosphoribosyltransferase (Lowy et al. (1980) “Isolation Of Transforming DNA: Cloning The Hamster aprt Gene,” Cell 22: 817-823) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) “Transformation Of Mammalian Cells With An Amplifiable Dominant-Acting Gene,” Proc. Natl. Acad. Sci. USA 77:3567-3570; O'Hare et al. (1981) “Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase,” Proc. Natl. Acad. Sci. USA 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) “Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase,” Proc. Natl. Acad. Sci. USA 78: 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev (1993) “Gene Therapy, Concepts, Current Trials And Future Directions,” Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) “The Basic Science Of Gene Therapy,” Science 260:926-932; and Morgan et al. (1993) “Human Gene Therapy,” Ann. Rev. Biochem. 62:191-217) and hygro, which confers resistance to hygromycin (Santerre et al. (1984) “Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells,” Gene 30:147-156). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al. (1981) “A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14.
The expression levels of polypeptides described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987). When a marker in the vector system expressing a polypeptide described herein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a polypeptide described herein or a polypeptide described herein, production of the polypeptide will also increase (Crouse et al. (1983) “Expression And Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes,” Mol. Cell. Biol. 3:257-266).
Once a polypeptide described herein has been recombinantly expressed, it may be purified by any method known in the art for purification of polypeptides, polyproteins or antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the polypeptide comprises an Fc domain (or portion thereof)), and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.
Other aspects of the present disclosure relate to a cell comprising a nucleic acid described herein or a vector described herein. The cell may be a prokaryotic or eukaryotic cell. In some embodiments, the cell in a mammalian cell. Exemplary cell types are described herein.
Yet other aspects of the disclosure relate to a method of producing a polypeptide described herein, the method comprising obtaining a cell described herein and expressing nucleic acid described herein in said cell. In some embodiments, the method further comprises isolating and purifying a polypeptide described herein.
Other aspects of the present disclosure relate to pharmaceutical compositions comprising the isolated polypeptides or the chimeric molecules described herein. The term “pharmaceutical composition”, as used herein, refers to the formulation of an isolated polypeptide described herein in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition can further comprise additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic agents).
In some embodiments, the pharmaceutical composition of the present disclosure comprise other therapeutic agents. In some embodiments, such other therapeutic agents comprise an additional isolated polypeptide fragment. In some embodiments, the additional isolated polypeptide fragment comprises the amino acid sequence of the cysteine-rich domain of FZD (FZD-CRD). Also illustrated in the Examples of the present disclosure, is the inhibitory effect of FZD-CRD on TcdB binding to cell surface FZDs via competition. By preventing TcdB from binding to FZDs, the FZD-CRD polypeptides not only block the entry of TcdB into the cells, but also prevent the inhibition of Wnt signaling by TcdB. Thus, further provided herein are examples of how the FZD-CRD polypeptides protect cells in from TcdB induced CDI. As illustrated herein, Triple FZD1/2/7 knockout (KO) cells were dramatically resistant to toxin entry. Furthermore, colonic organoids with reduced FZD1/2/7 were less sensitive to TcdB. Finally, FZD2-CRD prevented TcdB binding to colonic tissues in mice and the colonic epithelium in FZD7 KO mice was less susceptible to TcdB-induced tissue damage. These findings establish FZDs as physiologically relevant epithelial receptors for TcdB, point to a role of Wnt signaling blockage in CDI pathogenesis, and provide novel therapeutic targets for treating CDI. Recombinant human FZD-CRD proteins and variants are commercially available (e.g., from ACRO Biosystems).
In some embodiments, the additional isolated polypeptide fragment of the present disclosure, may comprise an amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the isolated polypeptide fragment comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 24. For example, the isolated polypeptide fragment comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to SEQ ID NO: 24. In some embodiments, the isolated polypeptide fragment comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 25. For example, the isolated polypeptide fragment comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to SEQ ID NO: 25. In some embodiments, the isolated polypeptide fragment comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 26. For example, the isolated polypeptide fragment comprises an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to SEQ ID NO: 26.
The additional isolated polypeptide fragments of the present disclosure, may comprise any modifications or derivatizations disclosed herein. Such additional isolated polypeptide fragments may also be fused to any heterologous partners described herein, e.g., an Fc domain.
As it may also become clear later in the present disclosure, the pharmaceutical composition of the present disclosure, may further comprise other therapeutic agents suitable for the specific disease such composition is designed to treat.
The term “pharmaceutically-acceptable carrier”, as used herein, means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the polypeptide from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
In some embodiments, an isolated polypeptide of the present disclosure in a composition is administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. Typically, when administering the composition, materials to which the polypeptide of the disclosure does not absorb are used.
In other embodiments, the isolated polypeptides of the present disclosure are delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled release systems are discussed, for example, in Langer, supra.
Isolated polypeptides of the present disclosure can be administered as pharmaceutical compositions comprising a therapeutically effective amount of a binding agent and one or more pharmaceutically compatible ingredients.
In typical embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human being. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.
The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. The polypeptides of the present disclosure can be entrapped in ‘stabilized plasmid-lipid particles’ (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757.
The pharmaceutical compositions of the present disclosure may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
In some embodiments, the isolated polypeptides described herein may be conjugated to a therapeutic moiety, e.g., an antibiotic. Techniques for conjugating such therapeutic moieties to polypeptides, including e.g., Fc domains, are well known; see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev., 62:119-158.
Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a polypeptide of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized polypeptide of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is an isolated polypeptide of the disclosure. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
The isolated polypeptides, chimeric molecules, and the pharmaceutical compositions comprising such isolated polypeptides of the present disclosure, may be used to treat a variety of diseases. In some embodiments, the diseases are caused, at least in part, by the dysregulation of Wnt signaling pathways. In some embodiments, the disease is Clostridium difficile infection. Thus, further provided herein are methods of treating Clostridium difficile infection, comprising administering to a subject in need thereof, a therapeutically effective amount of the isolated polypeptides or the pharmaceutical composition comprising such isolated polypeptides disclosed herein. The isolated polypeptides of or the pharmaceutical composition comprising such isolated polypeptides, is effective in blocking TcdB binding to FZDs.
In some embodiments, the pharmaceutically composition used for treating CDI of the present disclosure, further comprises additional therapeutic agents or polypeptides. For example, the isolated TcdB1114-1835 polypeptide fragment of the present disclosure, while being able to block the wild-type TcdB from entering the cells, still inhibits Wnt signaling due to its occupancy of the FZD receptors. Thus, agents that activate Wnt signaling downstream of the FZD receptors may afford additional therapeutic effects against CDI. Agents that activate Wnt signaling downstream of the FZD receptors are known in the art. Non-limiting examples of such agents include GSK-3 inhibitors such as Lithium (LiCl) and CHIR99021. GSK-3 inhibits Wnt signaling downstream of the FZD receptors. Therefore, GSK-3 inhibitors are able to activate Wnt signaling downstream of the FZD receptors. Other non-limiting examples of agents that induce Wnt signaling include, without limitation, SB 216763 (Tocris Bioscience, catalog #1616), BIO (Tocris Bioscience, catalog #3194), TCS 2002 (Tocris Bioscience, catalog #3869), TC-G 24 (Tocris Bioscience, catalog #4353), TWS 119 (Tocris Bioscience, catalog #3835), SB 415286 (Tocris Bioscience, catalog #1617), A 1070722 (Tocris Bioscience, catalog #4431), AR-A 014418 (Tocris Bioscience, catalog #3966), L803-mts (Tocris Bioscience, catalog #2256). The activating of Wnt signaling occurs in a cell. In some embodiments, the cell is a colonic epithelial cell.
In some embodiments, the pharmaceutically composition used for treating CDI of the present disclosure, further comprises an agent that inhibits the cysteine protease activity of TcdB. In some embodiments, the agent is ebselen. Ebselen (also called PZ 51, DR3305, and SPI-1005), is a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity. It acts as a mimic of glutathione peroxidase and can also react with peroxynitrite. Ebselen is a potent scavenger of hydrogen peroxide as well as hydroperoxides including membrane bound phospholipid and cholesterylester hydroperoxides. Several ebselen analogues have been shown to scavenge hydrogen peroxide in the presence of thiols. Ebselen is known in the art to be inhibiting the cysteine protease activity of TcdB. Other non-limiting examples of cysteine protease inhibitors include serpins, stefins, and Inhibitors of apoptosis (IAPs).
Yet in other embodiments, the pharmaceutically composition used for treating CDI of the present disclosure, further comprises agents that facilitate blocking TcdB binding to FZDs. Such agents may be, for example, an FZD antibody. It is to be understood that any agents that competes with TcdB for binding to FZD may be used herein.
In other embodiments, the disease caused by the dysregulation of Wnt signaling is cancer. The dysregulation of Wnt signaling pathway is a known cause of cancer and is a central mechanism in cancer biology. For example, Wnt overexpression could lead to malignant transformation of mouse mammary tissue. Therefore, the inhibition of Wnt signaling has been a focus for developing cancer therapeutics. As described herein, the isolated polypeptides of the present disclosure, e.g., the TdcB1114-1835 polypeptide, is able to inhibit/block Wnt signaling by competing with Wnt for the FZD receptors. Thus, other aspects of the present disclosure relate methods of treating cancer. Such methods comprise administering to the subject in need thereof a therapeutically effective amount of the isolated polypeptides, or the pharmaceutical composition comprising the isolated polypeptides of the present disclosure.
In some embodiments, the method of treating cancer of the present disclosure, further comprises administering to the subject an agent that blocks Wnt signaling. Non-limiting examples of agents that block Wnt signaling include Dkk family proteins, Secreted Frizzled Related Proteins (sFRP), Draxin, IGFBP-4, SOST/Sclerostin, USAG1, and WIF-1. In some embodiments, the agent that blocks Wnt signaling is an FZD antibody. The use of these agents in blocking Wnt signaling is known in the art.
Many types of cancer are characterized with over-activated Wnt signaling and over-expression of Frizzled. For instance, >90% of colon cancers feature aberrant Wnt signaling. Recent study (Gujral et al, Cell, 2014, 159, 844-856) showed that Frizzled 2 is over expressed in metastatic liver, lung, colon and breast cancers. The expression is highly correlated with the markers of epithelial-mesenchymal transition. Thus, types of cancer that may be treated using the methods disclosed herein include, without limitation neoplasms, malignant tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer may be a primary or metastatic cancer. Cancers include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma, teratomas, choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer. In some preferred embodiments, the methods of the present disclosure may be used to treat colon cancer, liver cancer, lung cancer, breast cancer. In some embodiments, the cancer cells are metastatic. It is to be understood that the examples are not meant to be limiting and that any types of cancer that shows hyperactive Wnt signaling or overexpression of Frizzled may be treated using the methods disclosed herein.
“A therapeutically effective amount” as used herein refers to the amount of each therapeutic agent of the present disclosure (e.g., the isolated polypeptide fragment, the additional isolated polypeptide fragment, and the agent that activates Wnt signaling) required to confer therapeutic effect on the subject, either alone or in combination with one or more other therapeutic agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual subject parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, therapeutic agents that are compatible with the human immune system, such as polypeptides comprising regions from humanized antibodies or fully human antibodies, may be used to prolong half-life of the polypeptide and to prevent the polypeptide being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a disease. Alternatively, sustained continuous release formulations of a polypeptide may be appropriate. Various formulations and devices for achieving sustained release are known in the art.
In some embodiments, dosage is daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the polypeptide used) can vary over time. In some embodiments, for an adult subject of normal weight, doses ranging from about 0.01 to 1000 mg/kg may be administered. In some embodiments, the dose is between 1 to 200 mg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular subject and that subject's medical history, as well as the properties of the polypeptide (such as the half-life of the polypeptide, and other considerations well known in the art).
For the purpose of the present disclosure, the appropriate dosage of a therapeutic agent as described herein will depend on the specific agent (or compositions thereof) employed, the formulation and route of administration, the type and severity of the disease, whether the polypeptide is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the antagonist, and the discretion of the attending physician. Typically the clinician will administer a polypeptide until a dosage is reached that achieves the desired result. Administration of one or more polypeptides can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a polypeptide may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a disease.
As used herein, the term “treating” refers to the application or administration of a polypeptide or composition including the polypeptide to a subject in need thereof. “A subject in need thereof”, refers to an individual who has a disease, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease. In some embodiments, the subject has CDI. In some embodiments, the subject has cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is human.
Alleviating a disease includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a disease includes initial onset and/or recurrence.
In some embodiments, the pharmaceutical composition comprising the therapeutic agents (e.g., an isolated polypeptide) described herein is administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of TcdB by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo or in vitro.
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
Genome-wide CRISPR/Cas9 Screen Reveals Frizzled as Receptors for Clostridium difficile Toxin B
To identify the physiologically relevant receptor and other host factors involved in TcdB actions, two unbiased genome-wide mutagenesis screens using the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 approach were performed (15, 16). The C-terminal part of TcdA and TcdB contains a region known as combined repetitive oligopeptides (CROPs,
HeLa cells that stably express RNA-guided endonuclease Cas9 were transduced with lentivirus libraries that express small guide RNA (sgRNA) targeting 19,052 genes, with six sgRNAs per gene (15). After four rounds of selection with increasing concentrations of toxins, the sgRNA sequences from the remaining cells were identified via next-generation sequencing (NGS). Candidate genes were ranked based on the number of unique sgRNAs identified for each gene (Y-axis) versus its total NGS reads (X-axis), which represents the abundance of cells harboring sgRNA targeting that gene (
UDP-glucose pyrophosphorylase (UGP2) stood out in both screens (
To validate the screening results, individual knockout HeLa cell lines for top candidates, including UGP2−/−, CSPG4−/−, FZD2−/−, and EMC4−/−, were generated using the CRISPR/Cas9 approach (
CSPG4/NG2 and FZD2 were investigated for their potential as receptors. Binding of TcdB to CSPG4−/− cells was drastically reduced and ectopic expression of rat NG2 restored binding (
Transfecting CSPG4−/− cells with full-length FZD2 also increased binding of TcdB (
In contrast to CSPG4, transfecting FZD2 in CSPG4−/− cells increased binding of both TcdB and TcdB1-1830 (
The CRDs of FZD1, 2, and 7 are highly conserved, with ˜98% sequence similarity and ˜84% identity (
As FZDs and CSPG4 are recognized by distinct regions of TcdB, the present data support a previously proposed two-receptor model for TcdB (19). Consistent with this model, FZD2-CRD binds robustly to TcdB that is pre-bound by immobilized CSPG4/NG2-EC on the micro-titer plate (
The role of FZDs and CSPG4 in human colorectal cell lines HT-29 and Caco-2, which express multiple FZDs was further examined (29). FZD2-CRD fully protected both cell types from TcdB1-1830 (
Whether FZDs are the pathologically relevant TcdB receptors in colonic epithelial cells was next examined. First, primary colonic organoid models, which develop into a “mini-gut” when cultured in 3-D matrix and display many important features of normal colonic epithelium, were used (30). Exposure to TcdB caused a concentration-dependent atrophy and death of organoids, which was quantified with a viability assay (
Wnt signaling plays a critical role for growth and survival of intestinal and colonic organoids. Both TcdB and Wnt bind to the FZD-CRD. It was found that a non-toxic fragment of TcdB (residues 1114-1835) potently blocked Wnt3a-mediated signaling in cultured cells, as demonstrated by the TOPFLASH luciferase reporter assay as well as phosphorylation levels of LRP6 and Dvl2, which are the FZD co-receptors and a downstream component, respectively (
The role of FZDs in vivo using mouse models was examined next. Because TcdB is naturally released into the lumen of the colon during CDI, a model was developed by injecting TcdB directly into the lumen of ligated colon segments in mice (
FZD2/7 double KO mice are embryonic lethal (25, 32). As FZD7 appears to be a dominant Wnt receptor in the intestinal epithelium (31), FZD7−/− mice were utilized as a model to determine whether depletion of FZD7 may reduce toxicity of TcdB on the colonic epithelium in vivo. To detect the damage to colonic tissues, TcdB1-1830 was injected directly into ligated colon segments of live mice, followed by an 8 hour incubation period. TcdB1-1830 was used instead of TcdB, in order to focus on the colonic epithelium and avoid complications from potential TcdB entry into CSPG4-expressing ISEMFs after the colonic epithelium is damaged. Accumulation of fluids was observed in the lumen of the ligated colon segments in the WT mice after exposure to TcdB1-1830, but was significantly reduced in that of FZD7−/− mice (
In addition to receptors, the screen also revealed other cellular factors, such as the EMC complex (
PVRL3 did not appear in the screens, which may not be surprising as PVRL3 was identified in a screen for factors involved in necrotic cell death induced by toxin concentrations several orders of magnitude higher than what was used in the present study to screen for cytopathic cell-rounding and apoptosis (14). The role of PVRL3 was examined experimentally and it was found that ectopically expressed PVRL3 did not mediate binding or entry of TcdB into CSPG4−/− HeLa cells (
The unbiased genome-wide CRISPR-mediated screens revealed multiple host factors involved in all major steps of toxin actions, from surface receptors (FZDs and CSPG4) to acidification in endosomes (vacuolar-type H+-ATPase), and to toxin enzymatic activities in the cytosol (UGP2). The screens also suggested EMC as a key factor for folding/trafficking of Wnt receptors. Interestingly, the screen identified a total of eleven proteins involved in Wnt signaling pathways, including APC, GSK-3β, Wnt5a, and LRP6 (
The present study showed FZDs are physiologically relevant receptors for TcdB in colonic epithelial cells, suggesting a potential new mechanism: TcdB may disrupt the colonic epithelium by directly blocking Wnt signaling. The present study also provided novel therapeutic targets for treating CDI. Furthermore, dysregulation of Wnt signaling pathways is associated with many cancers, particularly colorectal cancers. Therefore, the receptor binding domain of TcdB, or its homologs, are believed to be valuable tools and therapeutics for targeting Wnt pathways.
Materials and Methods
Cell Lines, Antibodies, and Constructs.
HeLa (H1), CHO (K1), HT-29, Caco-2, and HEK293T cells were obtained from ATCC. The following mouse monoclonal antibodies were purchased from indicated vendors: Rac1 (23A8, Abcam), non-glucosylated Rac1 (Clone 102, BD Biosciences), 1D4 tag (MA1-722, ThermoFisher Scientific), HA tag (16B12, Covance), β-actin (AC-15, Sigma). Rabbit monoclonal IgG against human CSPG4 (ab139406) and rabbit polyclonal antibodies against FZD1 (ab150553), FZD2 (ab150477), FZD7 (ab51049), PVRL3 (ab63931), and Claudin3 (ab15102) were all purchased from Abcam. Rabbit monoclonal antibodies against Dvl2 (30D2) and LRP6 (C5C7), and a rabbit polyclonal antibody against phosphorylated LRP6 (Ser1490) were all purchased from Cell Signaling. Chicken polyclonal IgY (#754A) against TcdB was purchased from List Biological Labs. A rabbit polyclonal antibody against rodent CSPG4/NG2 and a construct express full-length rat CSPG4/NG2 (in pcDNA vector) were both generated in W. Stallcup's lab. 1D4 tagged full length FZD1-10 constructs in pRK5 vectors were originally generated in J. Nathans's lab (Baltimore, Md.) and were obtained from Addgene. FZD7 and FZD8 CRD-myc-GPI constructs were generously provided by J. Nathan's lab and have been described previously (35). Constructs expressing full-length human IL1RAPL2 and full-length PVRL3 were purchased from Vigene Biosciences. A construct expressing full-length mouse Syt II in pcDNA3.1 vector was described previously (36).
TcdB and Other Recombinant Proteins.
Recombinant TcdB (from C. difficile strain VPI 10463) was expressed in Bacillus megaterium as previously described (37) and purified as a His6 tagged protein. TcdB1-1830 was cloned into the pHis1522 vector (MoBiTec) and expressed in Bacillus megaterium following the same procedure used for TcdB. TcdB1831-2366, TcdB1501-2366, and TcdB1114-1835 were cloned into pGEX-6P-1 or pET28a vectors and were purified as GST-tagged or His6-tagged proteins in E. coli. CSPG4/NG2 EC (P1 and P2) was expressed in HEK293 cells, purified from medium with DEAE-Sepharose columns, and eluted with a gradient buffer (NaCl from 0.2 to 0.8 M, 50 mM Tris-Cl, pH 8.6) as previously described (38). The following recombinant human proteins were purchased from ACRO Biosystems (IgG1 Fc and FZD2-CRD-Fc), R&D Systems (FZD1-CRD-Fc, FZDS-CRD-Fc, and FZD7-CRD-Fc), and Sino Biologics (PVRL3-EC).
Generating Stable HeLa-Cas9 Cells and Lentivirus sgRNA Libraries.
The human codon-optimized sequence of S. pyogenes Cas9 was subcloned from plasmid lentiCas9-Blast (Addgene #52962) into pQCXIH retroviral vector (Clontech), which was used to generate retroviruses to transduce into H1 HeLa cells (ATCC CRL-1958). Mixed stable cells were selected in the presence of 200 μg/ml hygromycin B (Life Technologies). Lentivirus sgRNA libraries were generated following published protocols using the human GeCKO v2 sgRNA library (Addgene #1000000049), which targets 19,052 genes in the human genome (15). The GeCKO v2 library is delivered from Addgene in two half-libraries (library A and library B). Each half library contains three unique sgRNA per gene and two half-libraries were subjected to screens with toxins independently. Cells were transduced with lentivirus-packaged GeCKO v2 sgRNA library at a MOI of 0.2.
Screening CRISPR libraries with TcdB and TcdB1-1830. For each half CRISPR library of cells, 4×107 cells were plated onto two 15-cm culture dishes to ensure sufficient coverage of sgRNAs, with each sgRNA on average being represented about 650 times (i.e., there are on average 650 cells transduced with the same sgRNA). This over-representation rate was calculated from titration plates that were set up in parallel with the library. These cells were exposed to either TcdB or TcdB1-1830, respectively, for 48 hours. Cells were then washed three times with PBS to remove loosely attached round-shaped cells. The remaining cells were re-seeded onto new dishes and cultured with normal media without toxins until the cells reach
Generating Knockout Cell Lines Via CRISPR.
The following sgRNA sequences were cloned into LentiGuide-Puro vectors (Addgene) to target indicated genes: ccggagacacggagcagtgg (cspg4) (SEQ ID NO: 3), gcgctgctgggacatcgcct (emc4) (SEQ ID NO: 4), accttataccacacaacatc (illrap12) (SEQ ID NO: 5), tgcgagcacttcccgcgcca (fzd2) (SEQ ID NO: 6), agcgcatgaccactacactg (sgms1) (SEQ ID NO: 7), acaggcagaaaacggctcct (ugp2) (SEQ ID NO: 8), GTGTAATGACAAGTTCGCCG (FZD1) (SEQ ID NO: 9), and GAGAACGGTAAAGAGCGTCG (FZD7) (SEQ ID NO: 10). HeLa-Cas9 cells were transduced with lentiviruses that express these sgRNAs. Mixed populations of stable cells were selected with 2.5 μg/ml puromycin (Gibco) and 200 μg/ml hygromycin B. Triple knockout cells of FZD1/2/7 were created by sequentially transducing FZD1 and 7 sgRNA lentiviruses into FZD2−/− cells, followed by selection with 50 pM TcdB1-1830. The knockout efficiency was demonstrated by NGS (
Cytopathic Assay.
The cytopathic effect (cell-rounding) of TcdB and TcdB1-1830 was monitored using well established standard cell-rounding assay as previously described (1). Briefly, cells were exposed to a gradient of TcdB and TcdB1-1830 added into media for 24 hours as shown in
Blocking TcdB Entry into Cells with Extracellular Domains of CSPG4/NG2 and FZD2.
Recombinant proteins used for cell protection assays were pre-filtered (0.22 μM filter, Millipore). Toxins were pre-incubated with FZD2-CRD-Fc and/or CSPG4-EC (P1) for 30 minutes on ice with a toxin:protein ratio of 1:400 except when noted in the figure legend. The mixtures were added into cell culture medium. The cytopathic effects were analyzed by cell-rounding assay as described above.
Transfection and Detection of TcdB Binding.
Transient transfection of HeLa cells was carried out with POLYJET™ transfection reagent (SignaGen) following the manufacturer's instruction. Binding of TcdB to cells was analyzed by exposing cells to TcdB or truncated TcdB fragments (10 nM, unless noted in the figure) for 10 min at room temperature, followed by washing three times with PBS. Cells were then either fixed and subjected to immunostaining, or harvested and subjected to immunoblot analysis.
GST Pull-Down Assays.
GST pull-down assays were performed using glutathione Sepharose 4B as previously described (36). Briefly, 5 μg of GST-tagged TcdB1831-2366 and TcdB1501-2366 were immobilized on glutathione beads and were incubated with 10 nM FZD2-CRD-Fc for one hour at 4° C. Beads were then washed, pelleted, and boiled in SDS sample buffers. Samples were subjected to immunoblot analysis.
Biolayer Interferometry Assay.
The binding affinities between TcdB and FZDs were measured by BLI assay with the Blitz system (ForteBio). Briefly, the CRDs-Fc of FZD1, 2, 5, 7 or human IgG1 Fc (20 μg/ml) were immobilized onto DIP AND READ™ Anti-hlgG Fc Capture Biosensors (ForteBio) and balanced with PBS buffer. The biosensors were then exposed to series concentrations of TcdB or TcdB1-1830, followed by washing with PBS. Binding affinities (KD) were calculated using the Blitz system software (ForteBio).
Wnt Signaling Assay.
The TOPFLASH/TK-Renilla dual luciferase reporter assay was utilized to detect Wnt signaling activities as previously described (39). Briefly, HEK 293T cells in 24-well plates were co-transfected with TOPFLASH (50 ng/well), TK-Renilla (internal control, 10 ng/well), and pcDNA3 (200 ng/well). After 24 hours, cells were exposed to Wnt3a (50 ng/ml) and TcdB1114-1835 (with molar ratio 1:8, 1:40, and 1:200 to Wnt3a, respectively) in culture medium for 6 hours. Cell lysates were harvested and subjected to the firefly/renilla dual luciferase assay, as well as immunoblot analysis detecting phosphorylated Dvl2 and LRP6. Wnt signaling activates expression of TOPFLASH luciferase reporter (firefly luciferase). Co-transfected renilla luciferase serves as an internal control.
Micro-Titer Plate Based Binding Assay.
Binding assays were performed on EIA/RIA Half Area 96-well plates (high-binding, Corning Costar) as described previously (38). Briefly, micro-titer plates were coated with 10 μg/ml CSPG4/NG2 proteins in coating buffer (0.1 M NaHCO3, pH 8.3) at 4° C. overnight, and then blocked with 1% bovine serum albumin in PBS for 1 hour. Plates were then incubates with the indicated proteins for 1 hour in PBS. Wells were washed three times with PBS plus 0.05% tween-20 at room temperature. One-step Turbo TMB (Thermo Scientific) was used as the substrate and absorbance at 450 nm was measured with a microplate reader.
Organoid Culture, Adenoviral Transduction, and TcdB Challenge Assay.
Crypt isolation from WT or FZD7−/− mouse colon was carried out as previously described and organoids were expanded as spheroid cultures using conditioned medium (40). Except for WT organoids used for Wnt-Signaling inhibition assay, 3 μM CHIR99021 was supplemented to the medium (31). Five days after passaging, organoids were re-suspended with Cell Recovery Solution (Fisher Scientific) and mechanically fragmented. Fragments were transduced with adenovirus expressing shRNA for FZD1, shRNA for FZD2, or a control sequence using transduction medium supplemented with Nicotinamide (10 mM, Sigma), Polybrene (8 ug/ml, Sigma) and Y-27632 (10 uM, Sigma), washed and plated in growth factor reduced Matrigel (Corning) (41). Three days following viral transduction, organoids were challenged with series diluted TcdB by directly adding the toxin into the medium. The MTT assay was performed to measure the viability of cells 72-hours post-exposure to the toxin.
Wnt Signaling Inhibition Assay in WT Colon Organoids.
TcdB1114-1835 of indicated concentration was directly added into the culture media of WT colon organoids. For rescue experiments, 5 μM CHIR99021 was added to the media. The media were changed every 48 hours with the constant presence of TcdB1114-1835 and CHIR99021. Viability of cells was analyzed after six days.
Adenovirus Mediated KD.
All shRNAs were purchased from sigma TRC shRNA designed library. The knockdown efficiency was validated as described in
Assessment of Viability of Colonic Organoids Using MTT Assay.
The viability of the organoids were assessed via the ability to reduce MTT as previously described (42). Briefly, MTT solution was added to the organoid culture to a final concentration of 500 μg/ml. After incubation at 37° C. for 2 hrs, the medium was discarded. For each well (20 μl of Matrigel, in 48-well plate), 60 μl of 2% SDS solution was added to solubilize the Matrigel (1 hour, 37° C.), followed by the addition of 300 μl of DMSO to solubilize reduced MTT (2 hours, 37° C.). The absorbance at 562 nm was measured on a microplate reader. Twenty μl of Matrigel without organoids were used as blank controls. Normal organoids without exposure to toxins were defined as 100% viable.
Immunohistochemistry (IHC) and Histology Analysis.
Colons from adult C57BL/6 mice (10-12 weeks old) were dissected out and subjected to cryosectioning with sections measuring 8-10 μm thickness. Colonic sections were fixed in cold acetone for 5 minutes and then washed three times with PBS. The colonic sections were then blocked with 5% goat serum in PBS for 30 minutes at room temperature, and incubated with primary antibodies (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250) overnight, followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 hour at room temperature. They were then incubated with HRP-conjugated streptavidin (1:500, DAKO) for 30 minutes. Immuno-reactivity was visualized as a red color with 3-amino-9-thyl carbazole (DAKO). Cell nuclei were labeled as a blue color with Gill's Hematoxylin (1:3.5, Sigma). Frozen human colon tissue slides were purchased from BioChain Institute Inc., and subjected to IHC analysis. IHC analysis of Claudin3 was carried out using mouse colon tissues fixed in 10% formalin and embedded in paraffin following standard procedures (anti-Claudin3 antibody: 1:100) and detected with 3-Amino-9-Ethylcarbazole (AEC). Histology analysis was carried out with H&E staining of paraffin-embedded sections. Stained sections were coded and scored by blinded observers based on disruption of epithelium, inflammatory cell filtration, and edema, on a scale of 0 to 3 (mild to severe).
Competition Assays in Colon Tissues with Recombinant Proteins.
TcdB (40 nM) was pre-incubated with either human IgG1-Fc or FZD2-Fc (2.4 μM) for 30 minutes on ice. To generate the ex vivo colon segment, mice (C57BL/6, 6-8 weeks) were euthanized and the colon was exposed via laparotomy. A segment in the ascending colon (˜2 cm long) was sealed by tying both ends with silk ligatures. The toxin samples (40 μl) were injected through a LV catheter into the sealed colon segment. The injection site was then sealed with a hemostat. The colon was covered with PBS-soaked gauze for 2 hours. The colon segment was then excised and its lumen was washed with PBS injected through a needle for three times, and then subjected to IHC analysis.
Colon Loop Ligation Assay.
All procedures were conducted in accordance with the guidelines of the Boston Children's Hospital IACUC. WT or FZD7−/− Mice (6-8 weeks) were anesthetized following overnight fasting. A midline laparotomy was performed to locate the ascending colon and seal a ˜2 cm long loop with silk ligatures. Two μg of TcdB1-1830 in 80 μl of normal saline or 80 μl of normal saline were injected through a LV catheter into the sealed colon segment, followed by closing the wounds with stitches. Mice were allowed to recover. After 8 hours, mice were euthanized and the ligated colon segments were excised out. The weight and length of ligated colon were measured and recorded. The colon segments were fixed and subjected to H&E staining and IHC.
Inhibition of Tumor Growth in Xenograft Models.
The effects of blocking Wnt signaling with TcdB1114-1835 on tumor growth is assessed in vivo using a well-established mouse xenograft model. Liver cancer cell lines FOCUS and Huh7 cells are used. These cells lines express high levels of FZD2 and inhibiting Wnt signaling by FDZ antibodies can reduce growth of tumors formed by these cancer cells in mouse xenograft models (Gujral T S et al. Cell, 2014, 159:844-856). FOCUS or Huh7 cells (2×106 in suspension) are inoculated subcutaneously (s.c.) into athymic nude mice on day 0. Tumor growth is followed every 2 to 3 days. The size of tumor is measured using Vernier calipers. The tumor volumes are calculated using the formula: V=AB2/2 (A, axial diameter; B, rotational diameter). When tumors reach ˜200 mm3, mice are divided into two groups (control and treatment). The treatment group are injected with TcdB1114-1835 (20 mg/kg in saline) subcutaneously at the tumor site twice a week for up to three weeks. The control group are injected with saline. The tumor size are measured every 2-3 days. Tumor tissues are dissected out and subjected to immunohistochemical analysis to evaluate the markers for Wnt signaling and cellular proliferation and activity (e.g. β-catenin, Ki67).
Significantly reduced tumor sizes are observed in treated group than the control group, demonstrating that blocking Wnt signaling using TcdB1114-1835 inhibited tumor growth in vivo.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
This application claims the benefit of the filing date under 35 U.S.C. § 119 of U.S. Provisional Application Ser. No. 62/311,381, filed Mar. 21, 2016, and entitled Compositions and Methods for Inhibiting WNT Signaling, the entire contents of which are incorporated herein by reference.
This disclosure was made with government support under grant 1R01NS080833, awarded by the National Institutes of Health. The government has certain rights in the disclosure.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/23381 | 3/21/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62311381 | Mar 2016 | US |
