专利汇可以提供CBL-B INHIBITOR COMPRISING A CBL-B BINDING PEPTIDE FOR THE PREVENTION OR TREATMENT OF FUNGAL INFECTIONS专利检索,专利查询,专利分析的服务。并且The present invention relates to the prevention or treatment of fungal infections. The present invention provides a Cbl-b inhibitor for use in the prevention or treatment of a fungal infection in a patient, a Cbl-b inhibitor comprising a binding peptide of Syk, a fusion peptide comprising a cell delivery portion and a Cbl-b binding peptide, wherein the Cbl-b binding peptide binds to the TKB domain of Cbl-b, kits and uses for the same.,下面是CBL-B INHIBITOR COMPRISING A CBL-B BINDING PEPTIDE FOR THE PREVENTION OR TREATMENT OF FUNGAL INFECTIONS专利的具体信息内容。
The present invention relates to novel Cbl-b inhibitors and the inhibition of Cbl-b to modulate the activity of the immune system, especially in the prevention or treatment of fungal infections, in particular candidiasis, or cancer.
The E3-ubiquitin-ligase Cbl-b (casitas b-lineage lymphoma-b) has originally been described as a negative regulator of the adaptive immune response. Both T cell receptor (TCR) signaling and B cell receptor (BCR) signaling are modulated by Cbl-b mediated ubiquitination of critical signaling molecules. Consequently Cbl-b has been a target for immune therapy in the treatment of several diseases, especially cancer (
Fungi are increasingly recognized as major pathogens in critically ill patients (Enoch et al., 2006) and fungal infections claim an estimated 1.5 million lives every year (Brown et al., 2012). Candida species and Cryptococcus species are the yeasts most frequently isolated in clinical practice. Candida albicans is the most frequent fungal pathogen isolated from patients suffering from severe forms of fungal infections in the developed world (Kullberg et al., 2015). Aspergillus species are the most commonly isolated invasive molds (Denning, 1998). The use of antineoplastic and immunosuppressive agents, broad-spectrum antibiotics, prosthetic devices and grafts, and more aggressive surgery are reasons for the increase in invasive fungal infections. Patients with burns or pancreatitis are also predisposed to fungal infection (Enoch et al., 2006). Patients with an impaired immune status, as observed upon HIV infection (Armstrong-James et al., 2014; Brown et al., 2014; Park et al., 2009), immune suppression (Ravikumar et al., 2015), or certain types of primary immune deficiencies (Lanternier et al., 2013; Lortholary et al., 1997) are at high risk of developing a severe fungal infection.
Yeasts, molds and dimorphs are three types of fungi known to cause disease in humans (Jain et al., 2010). Fungal infections are classified into 3 groups: 1) superficial/cutaneous infections, present on skin, hair and nails, 2) subcutaneous infections, present in tissues under the skin and 3) systemic infections.
Several anti-fungal agents are used for the treatment of the different kinds of fungal infections. Amphotericin B, a polyene with a very broad spectrum of activity, including most yeasts and molds, belongs to the class of polyene anti-fungals and can be administered locally or systemically. However, side effects are occurring in 50-90 % of cases, and are usually nephrotoxicity or infusion-related. Fever, chills, nausea, vomiting and hypotension are further side effects. Fluconazole belongs to the class of azole anti-fungals and is administered orally or intravenously. Disadvantages include resistance of several Candida strains and adverse effects such as headache, nausea, vomiting and elevated liver enzymes.
Taken together, efficacy and/or safety of known anti-fungal agents are still lacking in many circumstances and there is a significant need for new anti-fungal therapies. It is one of the objects of the present invention to provide novel compounds and methods for preventing or treating fungal infections.
The present invention provides methods and means for inhibition of casitas b-lineage lymphoma-b (Cbl-b) activity, which can significantly ameliorate patients health when diseased or infected, in particular by fungal infections, most strikingly by significantly reducing morbidity (see e.g.
In another aspect of the invention, a fusion peptide comprising a cell delivery portion and a Cbl-b binding peptide, wherein the Cbl-b binding peptide binds to the tyrosine kinase binding (TKB) domain of Cbl-b, is provided. Notably, this fusion peptide has turned out to be very effective in protecting from lethal fungal infections (
Moreover, the invention relates to a fusion peptide comprising a cell delivery portion and a Cbl-b binding peptide, wherein the Cbl-b binding peptide is an intramer or aptamer, and wherein the Cbl-b binding peptide inhibits Cbl-b. Preferably, the Cbl-b binding peptide binds to the TKB domain of Cbl-b.
The present invention further relates to a Cbl-b inhibitor for use in a patient as a vaccine adjuvant, wherein the Cbl-b inhibitor is a Cbl-b binding peptide. Preferably, the Cbl-b binding peptide binds to the TKB domain of Cbl-b. Especially preferred it is a peptide (fragment) of or derived from Syk.
The present invention also relates to a pharmaceutical composition, comprising the fusion peptide or the Cbl-b binding peptide, and a pharmaceutically acceptable carrier promoting cellular uptake of said peptide.
Even further, the invention provides a vaccine, comprising as an adjuvant a Cbl-b inhibitor. Preferably, the Cbl-b inhibitor is a Cbl-b binding peptide of Syk, and at least one antigen.
In another aspect, the present invention also relates to a kit comprising said peptide and a pharmaceutically acceptable excipient. Preferably, the pharmaceutically acceptable excipient is suitable for intracellular administration in the patient.
The present invention also relates to a method for regulating innate immunity, comprising the inhibition of Cbl-b. Preferably, said fusion peptide or Cbl-b inhibitor are administered to the patient.
Moreover, in another aspect, the present invention relates to an in vitro or in vivo method for stimulating or activating an immune cell, comprising contacting or treating the cell with said fusion peptide or Cbl-b binding peptide.
All aspects and embodiments of the invention relate to the inhibition of Cbl-b using a Cbl-b binding peptide. All general and specific descriptions provided herein in more detail, including preferred embodiments relate to all aspect and uses of the invention equally, except where explicitly stated. E.g. any preferred definition of the peptide or its Cbl-b binding portion can be used in any aspect or method of the invention.
The E3-ubiquitin-ligase Cbl-b is a member of the Cbl family of proteins, playing key roles in regulating signaling in a variety of cellular systems (Sohn et al., 2003). The amino-terminal domain of Cbl proteins is composed of a tyrosine kinase binding (TKB) domain and a RING finger domain, which are both modulating signal transduction and protein degradation (Ohno et al., 2016; Peschard et al., 2004). Substrate recognition by Cbl-b is mainly mediated by its TKB domain, which recognizes phosphotyrosines on target molecules, such as ZAP-70, Syk and Src and with residues located in RTKs such as EGFR, Met and CSF-1 receptors (Mancini et al., 2002; Peschard et al., 2001; Peschard et al., 2004; Tsygankov et al., 2001). Cbl-b has originally been characterized as a negative regulator of the adaptive immune response (Bachmaier et al., 2000; Chiang et al., 2000). Both T cell receptor (TCR) signaling and B cell receptor (BCR) signaling are modulated by Cbl-b mediated ubiquitination of critical signaling molecules (Loeser et al., 2007; Kitaura et al., 2007). Genetic ablation of Cbl-b (Bachmaier et al., 2000; Chiang et al., 2000) or knock-in of a catalytically inactive Cb1-bc373A/c373A variant (Paolino et al., 2011) has thus been reported to cause broad autoimmune manifestations, particularly after antigenic challenge. Recent observations uncovered additional roles for Cbl-b in NK cell-mediated anti-tumor immunity (Paolino et al., 2014), FcεR signaling in mast cells (Gustin et al., 2006; Qu et al., 2004), integrin signaling (Han et al., 2010), and DC functions (Wallner et al., 2013), extending the range of Cbl-b functions to cells of the innate immune system. Signaling molecules targeted by Cbl-b are often not specific to defined pathways, but can be used by multiple signaling modules.
Spleen tyrosine kinase (Syk) is a signaling protein targeted by Cbl-b. Canonical signaling induced by, amongst others, the C-type lectin receptors (CLRs) Dectin-1, -2, and -3 is critically dependent on activation of Syk (Gross et al., 2009; Mocsai et al., 2010). Syk is recruited to Dectin-1, Dectin-2/3, and Mincle receptor complexes and subsequently activated by both phosphorylation via Src family kinases and auto-phosphorylation (Deng et al., 2015). Activated Syk then triggers the inflammasome (Gross et al., 2009; Gringhuis et al., 2012; Zwolanek et al., 2014), canonical NF-κB signaling (LeibundGut-Landmann et al., 2007; Ruland, 2008), and production of reactive oxygen species (ROS) in monocytes, macrophages, neutrophils and dendritic cells (DCs) (Mocsai et al., 2010). Collectively, the activation of Syk by CLRs causes an immediate activation of fungicidal pathways, as well as inducing cell type specific transcriptional changes tailored to eradicating local or systemic fungal infections.
According to the invention, it was found that administration of a Cbl-b inhibitor protected from morbidity and mortality induced by infection with Candida albicans, the most prevalent yeast pathogen, and similar fungal pathogens. Thus, in a first aspect, the present invention provides a Cbl-b inhibitor for use in the prevention or treatment of a fungal infection in a patient, wherein the fungal infection is a yeast infection. The patient may be in need of a therapy, e.g. having disease symptoms or an adverse prognosis.
The Cbl-b gene and its gene products are well described in the art (see for instance also UniGene Id. Mm.328206 and Hs.430589). Cbl-b sequences are published e.g. in GenBank database under acc. no. NP_001028410.1 (Mus musculus) and NP_733762.2 (Homo sapiens). Cbl-b inhibitors are known in the art. Anti-Cbl-b antibodies, siRNAs and antisense inhibitors suitable as Cbl-b inhibitors are commercially available. Specifically, siRNAs suitable for reduction or inhibition of Cbl-b expression and thus also of Cbl-b function are disclosed for example in
Any Cbl-b inhibitor can be used according to the present invention. In one embodiment, the Cbl-b inhibitor is selected from nucleic acids targeting Cbl-b, such as Cbl-b inhibiting nucleic acids, siRNA, shRNA, miRNA.
It has been shown that the immune response to several clinically relevant yeast infections such as Candida infections of the family Saccharomycetaceae are regulated by Cbl-b. Thus, in a preferred embodiment, the yeast infection is of a yeast of the family Saccharomycetaceae, more preferably a candidiasis or a cryptococcosis. More preferably, the fungal infection is a Candida albicans, Candida glabrata, Candida krusei or a Candida dubliniensis infection.
The present invention also relates to a Cbl-b inhibitor for use in the prevention or treatment of a fungal infection in a patient, wherein the Cbl-b inhibitor is a Cbl-b binding peptide of Syk. The Syk gene and its gene products are well known in the art (UniGene Id. Mm.375031, Rn.87407, Mmu.32294 and Hs.371720). Syk sequences are published for example in GeneBank database under acc. nr. NP_001185906.2 (Mus musculus), NP_036890.1 (Rattus norvegicus), NM_001261035.1 (Macaca mulatta) and NP_003168.2 (Homo sapiens). Fragments of Syk that contain the sequence responsible for binding to Cbl-b (Cbl-b binding peptide) have been shown to be a particularly effective treatment agent for the therapeutic inhibition of Cbl-b. In particular, the Cbl-b-binding peptide binds to the TKB domain of Cbl-b (a TBK (domain) binding peptide).
In a preference, the Cbl-b binding peptide comprises the phosphotyrosine and surrounding flanking regions of Syk (
In an especially preferred embodiment of the invention, the Cbl-b binding peptide comprises the following consensus sequence: STVSFNPYEPX1X2X3X4WX5 (SEQ ID NO: 5), wherein X1, X2, X3, X4 and X5 are selected independently from any amino acid, preferably containing proteinogenic and/or non-proteinogenic amino acids as described above. In mice, the homologous sequence is STVSFNPYEPTGGPWG (SEQ ID NO: 6) in position 310-325 of Syk (Syk310-325). In rats, the homologous sequence is STVSFNPYEPTGGAWG (SEQ ID NO: 7) in position 310-325 of Syk (Syk310-325). In rhesus monkeys, the homologous sequence is STVSFNPYEPEVAPWA (SEQ ID NO: 8) in position 316-331 of Syk (Syk316-331). In humans, the homologous sequence is STVSFNPYEPELAPWA (SEQ ID NO: 9) in position 293-308 of Syk (Syk293-308). Thus, in a related embodiment, the Cbl-b binding peptide comprises, preferably consists of, the sequence STVSFNPYEPTGGPWG (SEQ ID NO: 6), STVSFNPYEPTGGAWG (SEQ ID NO: 7), STVSFNPYEPEVAPWA (SEQ ID NO: 8) or STVSFNPYEPELAPWA (SEQ ID NO: 9). Especially, the tyrosine (y) in the indicated sequence portion -PYE- in the disclosed sequences is phosphorylated. Preferably X1 is T or E. Preferably X2 is G, L or V. Preferably, X3 is G, or A. Preferably, X4 is P or A. Preferably, X5 is G or A. Also variants of these sequences are possible, therefore, the invention also relates to providing or using peptides comprising a sequence selected from STVSFNPYEPX1X2X3X4WX5 (SEQ ID NO: 5), STVSFNPYEPTGGPWG (SEQ ID NO: 6), STVSFNPYEPTGGAWG (SEQ ID NO: 7), STVSFNPYEPEVAPWA (SEQ ID NO: 8) or STVSFNPYEPELAPWA (SEQ ID NO: 9) with one, two or three amino acid deletions or substitutions at any position.
Preferably the Cbl-b binding peptide (or its portion in the fusion peptide) is a short peptide with 5 to 100 amino acids in length, preferably 8 to 80 or 12 to 60 amino acids in length.
The entire fusion peptide in any embodiment can be a peptide with 15 to 500 amino acids in length, preferably 15 to 200 amino acids in length, even more preferred 18 to 100 amino acids in length.
The Cbl-b binding peptides that bind to the TKB domain of Cbl-b, thereby inhibiting Cbl-b activity can be administered to a cell in any form, conjugate or composition. These forms, conjugates or compositions for administration allow entry of the Cbl-b peptide into a living cell, thereby passing a cell membrane. Preferred are conjugates with cell delivery or cell penetrating moieties, such as fusion peptides with cell penetrating peptides. Conjugates and fusion peptides may be connected by a linker moiety or linker peptide. Such linkers are usually small, e.g. less than 10kDa, or less than 5 kDa, or in the range of 0, or 1 to 20 amino acids in length. Other forms for cellular administration include vesicles, micelles, liposomes or microsomes.
Interestingly, a fusion peptide binding to the TKB domain of Cbl-b (
Cell penetrating peptides (CPPs) are short peptides, usually cationic or amphipathic, that mediate the intracellular delivery of a variety of biological cargos (Bechara et al., 2013; Jones et al., 2005, en.wikipedia.org/wiki/Cell-penetrating_peptide, all incorporated herein by reference). Peptides derived from proteins, chimeric peptides and synthetic CPPs are the three main classes of CPPs that can be distinguished (Bechara et al., 2013). Having important advantages such as low toxicity, CPPs have become common biotechnological tools for cross-cellular transport of biologically active peptides in vitro and in vivo (Jones et al., 2005; Lindsay et al., 2002). Thus, in a preferred embodiment, the cell delivery portion of the fusion peptide is a cell penetrating peptide. Important representatives of CPPs are Antennapedia, Tat, Transportan, dNP1, dNP2 (Lim et al., 2015) or Polyarginine. Antennapedia has been shown to be especially useful for in vitro peptide delivery, taking into account both the magnitude of uptake and cytotoxicity (Jones et al., 2005). Especially effective are CPPs comprising two, three, four, five, six, seven or more cations, especially cationic amino acid residues, such as H, K or R, preferably selected from K or R. Preferably, the CPP is selected from DRQIKIWFQNRRMKWKK (SEQ ID NO: 10), GRKKRRQRRRPPQ (SEQ ID NO: 11), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 12), (KKDKKDERRXAK)m (SEQ ID NO: 13), wherein 1≤m≤5 and XA is selected from K or R, and (KIKKXBKKKGRK)m (SEQ ID NO: 14), wherein 1≤m≤5 and XB is selected from V, I or T, or (R)n, wherein 6≤n≤12. Preferably the CPP (or its portion in the fusion peptide) is a short peptide with 8 to 100 amino acids in length, preferably 10 to 80 or 12 to 60 amino acids in length.
In another preferred embodiment, the Cbl-b binding peptide of the fusion peptide is of or derived from Syk, as described above. Preferably, the Cbl-b binding peptide is of Syk and the peptide is a regulator of innate immunity. The peptide is preferably a regulator of innate anti-parasitic immunity or anti-fungal immunity.
Other proteins or peptides binding to the TKB domain of Cbl-b can be used, such as proteins or Cbl-b binding peptides of the insulin receptor substrate 1 (IRS-1), a key intermediate of skeletal muscle growth regulated by insulin-like growth factor (IGF-1) (Nakao et al., 2009; Ohno et al., 2016) or the Zeta-associated protein of 70 kDa (Zap-70), playing a role in T-cell development and lymphocyte activation. In addition, residues located in receptor tyrosine kinases (RTKs) such as EGFR, Met and CSF1 receptors that interact with the TKB domain of Cbl-b can be used (Mancini et al., 2002; Nakao et al., 2009; Ohno et al., 2016; Peschard et al., 2001; Peschard et al., 2004; Tsygankov et al., 2001). Some of the TKB binding sites of Cbl-b share a sequence motif comprising the phosphotyrosine. Thus, the Cbl-b binding peptide preferably has the sequence motif N/DXpY, wherein X is selected from any amino acid, preferably containing proteinogenic and/or non-proteinogenic amino acids as described above. N/D is either asparagine (N) or aspartic acid (D), and pY is phosphotyrosine. More preferably, the Cbl-b binding peptide has the sequence motif N/DX1pYX2P (SEQ ID NO: 15), wherein X1 and X2 are selected from any amino acid, preferably containing proteinogenic and/or non-proteinogenic amino acids as described above. N/D is either asparagine (N) or aspartic acid (D), and pY is phosphotyrosine. Even more preferred, the Cbl-b binding peptide has a sequence length of 5 to 20 amino acids and/or a sequence identity to Syk of at least 80%, preferably at least 90%. In a further embodiment, the Cbl-b binding peptide is a fragment of Zap-70, or IRS-1. In Zap-70 of mice und humans, the homologous sequence of the sequence motif N/DX1pYX2P (SEQ ID NO: 15) is DgpYTP (SEQ ID NO: 16) in position 288-292 in mice (mouse Zap-70288-292) and in position 290-294 in humans (human Zap-70 290-294). In IRS-1 of mice and humans, the homologous sequence of the sequence motif N/DX1pYX2P is DgpYMP (SEQ ID NO: 17) in position 606-610 in mice (mouse IRS-1606-610) and in position 610-614 in humans (human IRS-1610-614). Thus, in a further preferred embodiment, the Cbl-b binding peptide comprises or consists of DGpYTP or DgpYMP.
Preferably, the Cbl-b binding peptide comprises a tyrosine, wherein the tyrosine is phosphorylated. Phosphorylating the tyrosine increases interaction with Cbl-b and is a preferred embodiment in all aspects of the invention.
The invention further relates to a pharmaceutical composition, comprising the fusion peptide or the Cbl-b binding peptide, and a pharmaceutically acceptable carrier. The carrier may promote cellular uptake of said peptide.
In one preferred embodiment, the pharmaceutically acceptable carrier is selected from vesicles, micelles, liposomes and microsomes. These may increase cellular uptake of the peptide, for therapeutic or also non-therapeutic uses. Pharmaceutical compositions can comprise pharmaceutically acceptable salts, buffers, tonicity agents or pharmaceutically acceptable carriers. Pharmaceutical carriers provide improved compatibility of the composition and enable improved solubility as well as improved bioavailability of the active ingredients. Examples are emulsifiers, thickening agents, redox components, starch, alcohol solutions, polyethylene glycol or lipids. In another preferred embodiment, the pharmaceutical composition is prepared or used for parenteral administration, especially intravenous, intraarterial, intramuscular, intrathecal, subcutaneous or intraperitoneal administration. In another preferred embodiment, the pharmaceutical composition is prepared or used for topical administration, preferably further comprises a skin penetration enhancer. Suitable skin penetration enhancers are selected from the group comprising C12-C15 alkyl benzoate, pentylene glycol, polyethylene glycol, ethoxydiglycol, dimethyl sulfoxide, sodium lauryl sulfate, polysorbate polyethylene sorbitan monolaurate, lecithin, and mixtures thereof.
Preferably, the peptide or pharmaceutical composition is for use in therapy, wherein the peptide or composition is administered to a patient. More preferably, the peptide or pharmaceutical composition is for use in the prevention or treatment of a fungal infection in the patient.
Toll-like receptors (TLRs) and CLRs have been shown to recognize conserved structures of invading pathogens, termed pathogen-associated molecular patterns (PAMPs), and thus induce immune responses (Kawai and Akira 2010; Kawai and Akira, 2011). Chiba et al. (2014) demonstrated that the CLR Dectin-1 recognizes tumor cells and, as a consequence, activates innate immune cells for anti-tumor responses. Dectin-1 serves as receptor for glyco-epitopes specifically expressed by tumor cells. Due to the function of Cbl-b in negative regulation of proximal Dectin-1 signaling, the TKB-binding peptide enhances Dectin-1 signal transduction impacting on the immune recognition of tumor cells by the innate immune system. In addition to the increase in activation of the immune system by the inventive inhibition of Cbl-b, this mechanism further enhances anti-tumor immunity driven by the innate immune system. Therefore, in a further preferred embodiment of the invention, the peptide or pharmaceutical composition is for use in the prevention or treatment of tumors, cancer, viral infections, bacterial infections, parasitic infections, especially intracellular parasites, mycosis, or drug-induced immunosuppression in a patient. Further disease or conditions to be treated are characterized by a congenital or acquired immune insufficiency, such as AIDS, multiple myeloma, chronic lymphatic leukemia, drug-induced immunosuppression or a cancer, optionally with the selection of disease-specific antigens. Treatment of a cancer involving solid tumors is preferred in particular.
N-glycan structures are highly expressed in tumor cells. In another preferred embodiment of the invention, the peptide or pharmaceutical composition is for use in the prevention or treatment of such tumors, wherein the tumor cells express N-glycan at high amounts. High levels of N-glycan amounts (molar) in a particular cell type are at least 1.5-fold of the N-glycans found in said non-tumor cell type. These non-tumor cell types are e.g. melanocytes, epithelial cells, lymphatic cells, mesenchymal cells, lung cells, blood cells, liver cells. According to preferred embodiments of the invention, the cancer or tumor disease to be treated according to the present invention is selected from ovarian cancer, testicular cancer, prostate cancer, breast cancer; cancer diseases of the digestive tract, in particular stomach cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer and liver cancer; kidney cancer, skin cancer, in particular melanoma, basal cell carcinoma and squamous cell carcinoma; neuroblastoma and glioblastoma, lung cancer, thyroid cancer, sarcoma, head and neck cancer, squamous cell carcinoma, lymphoma and leukemia (wherein the terms "cancer", "tumor", "carcinoma" etc. are always used interchangeably herein and refer to malignant diseases).
Preferably, the peptide or pharmaceutical composition for use is administered to the patient by intraperitoneal injection, comprising 1 mg to 500 mg of the fusion peptide or Cbl-b binding peptide, preferably for up to 7 days, 14 days, 21 days or 28 days and/or daily. Preferred doses are also 5 mg to 200 mg and 10 mg to 100 mg.
Cbl-b is a negative regulator of the innate and adaptive immune system (
"Vaccination" as used herein is not to be understood in the absolute sense - i.e., administration of an immunogen which leads to absolute protection by the immune system - but rather as immunological administration to increase protection by the immune system and/or to activate the immune system, in particular the cells thereof against the vaccine antigen. The inventive Cbl-b inhibitor, especially the inventive peptide, increase the immunoreactivity to a vaccine antigen in a patient and/or increasing the immunoreactivity per se in patient.
In a preferred embodiment, the patient to be treated is a mammal. In an even more preferred embodiment, the patient is a human. In another embodiment, the Cbl-b inhibitor for use as a vaccine adjuvant is administered to the patient together with the vaccine that comprises the antigen against which an immune reaction shall be increased by Cbl-b inhibition. In an alternative embodiment, the Cbl-b inhibitor is administered to the patient before and/or after administration of the vaccine.
It has been shown by the present invention that inhibition of Cbl-b is effective in the treatment of both systemic and cutaneous fungal infections, preferably candidiasis (e.g. example 3,
Preferably, the patient has or is at risk of developing sepsis, especially sepsis due to a fungal infection ("fungal sepsis"). The inventive increase in immune stimulation, especially of immune reactions against pathogens, may prevent or reduce the risk of sepsis. In an acute sepsis, it can quicken immune clearance.
The disease or condition to be treated may be acute or chronic. Chronic conditions are defined to last at least 90 days. Acute treatment may commence on days 0 to 89 after onset of the disease or condition, preferably on days 4 to 30.
In preferred embodiments of the present invention, the reduction or inhibition of the function of Cbl-b is transient, i. e. the function is only temporarily reduced and can therefore recover again, e. g. by consumption or degradation of inhibitors. The transient reduction of Cbl-b in immune cells in vitro or in a patient can also be performed in a repetitive manner, e. g. until a therapeutic success has been achieved.
In a further preferred embodiment of the invention, the Cbl-b inhibitor or peptide or pharmaceutical composition is administered to the patient intravenously, intraarterially, intramuscularly, intravascularly, subcutaneously, or intraperitoneally. More preferably, the patient to be treated is a mammal. Even more preferably, the patient is a human.
In a further preference, the Cbl-b inhibitor or peptide for use is administered to the patient using a pharmaceutically acceptable carrier, especially vesicles, micelles, liposomes or microsomes.
The present invention therefore also relates to a method for regulating, especially increasing, innate immunity, comprising the inhibition of Cbl-b at least in immune cells, e.g. in a patient as described above. Preferably, a fusion peptide or Cbl-b binding peptide as disclosed herein are administered to the patient. More preferably, the innate immunity is innate anti-parasitic immunity, innate anti-fungal, or innate anti-tumor immunity.
Immune cells targeted or affected by the inventive treatment are preferably antigen-presenting cells, PBMCs (peripheral blood mononuclear cells), T-lymphocytes, B-lymphocytes, monocytes, macrophages and/or dendritic cells, in particular T-lymphocytes, CD8+ T-lymphocytes, CD4+ T-lymphocytes, in particular Th1, Th2, Th17, Tregs (regulatory T-cells) or CTL (cytotoxic T-cells) or NK cells.
The present invention further relates to a kit comprising the peptide and a pharmaceutically acceptable excipient. Preferably, the kit is suitable for intracellular administration in the patient. Any route of administration to the patient as described above may be selected. The kit may comprise a container comprising the compounds, e.g. peptides or Cbl-b inhibitor of the invention and auxiliary substances of a pharmaceutical composition or other reagents to treat cells, the kit may be used in any method or any uses of the invention. The container and/or the kit may also comprise a further immunostimulating substance (apart from the peptide of the invention), preferably cytokine(s) or ligands of other receptors (e.g., TLRs) or antibodies to surface molecules, preferably CD3 and/or CD28, to additionally enhance the stimulation. Likewise, the kit or the container may also comprise stabilizing components, media or buffers.
In the kit or in a combinatory treatment of the patient or of cells, in vitro or in vivo, the Cbl-b inhibitor of the invention may be combined with further immune stimulating compounds such as an immune cell-stimulatory cytokine, e. g. a cytokine selected from the common gamma-chain cytokines, in particular IL-2, IL-15 and IL-21; cytokines that stimulate both the cells of the adaptive and of the innate immune system, in particular IL-12, IL-23 and IL-27; effector cell cytokines, such as IL-1, IL-17 and IL-18; an interferon, in particular interferon-alpha; or an interferon stimulator; an antibody, in particular an antibody which recognizes tumor cell surface molecules or a TLR or PAMP receptor ligand, in particular agonists, preferably of TLR-1, TLR-2, TLR-3, TLR-7, TLR-8 and TLR-9. An immune cell-stimulatory cytokine, a cytokine of both the adaptive and the innate immune system, an effector cell cytokine or an interferon stimulator according to the present invention are preferably selected from the group comprising IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17a, IL-17f, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, TNF (formerly TNF-alpha) and Lymphotoxin-alpha (formerly TNF-beta). The terms "simultaneous" or "together with" or "in combination with" or "combined with" as used in the context of the administration of the substances according to the present invention refer to the administration of at least one immune stimulating compound other than the Cbl-b inhibitor. If the administration is carried out using a plurality of different pharmaceutical compositions, the coadministration may be conducted simultaneously or sequentially.
Cbl-b acts in innate immune cells, especially in macrophages and dendritic cells, in vitro and that inhibition of Cbl-b increased immune response or activity of immune cells. Thus the invention relates to activating immune cells. The immune cells may be antigen-presenting cells, PBMCs (peripheral blood mononuclear cells), T-lymphocytes, B-lymphocytes, monocytes, macrophages and/or dendritic cells, in particular T-lymphocytes, CD8+ T-lymphocytes, CD4+ T-lymphocytes, in particular Th1, Th2, Th17, Tregs (regulatory T-cells) or CTL (cytotoxic T-cells) or NK cells. Such cells can also be treated and stimulated with the inventive Cbl-b inhibitor. The present invention therefore provides an in vitro method for stimulating or activating an immune cell, comprising contacting or treating the cell with a fusion peptide or a Cbl-b binding peptide as disclosed herein. Preferably, the method comprises the production of cytokines by the activated or stimulated immune cells. E.g., the produced cytokines are selected from IL-6, IL-1β, IL-23 and TNF.
The term "treating" or "treatment" as used herein means to cure an already present disease state or condition or to increase the likelihood of recovery from the disease state or condition. Treating can also include inhibiting, i.e. arresting the development of a disease state or condition, and ameliorating, i.e. causing regression of a disease. Treatment can be to ameliorate disease symptoms without curing a patient.
The term "preventing" or "prevention" as used herein does not mean to stop a disease state or condition from occurring in a patient or subject completely but may also refer to a reduced risk of developing a disease state or condition.
In a preference, the pharmaceutical composition of the present invention can further comprise one or more excipients pharmaceutically acceptable for parenteral administration. Suitable excipients are known to the person skilled in the art, for example water (especially water for injection), saline, Ringer's solution, dextrose solution, buffers, Hank solution, vesicle forming compounds (e.g. lipids), fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. This pharmaceutical composition can (as a drug) be administered via appropriate procedures known to the skilled person to a patient in need thereof (i.e. a patient having or having the risk of developing the diseases or conditions mentioned herein). The preferred route of administration of said pharmaceutical composition is parenteral administration, especially through intravenous, intraarterial, intramuscular, intrathecal, subcutaneous or intraperitoneal administration. For parenteral administration, the pharmaceutical composition of the present invention is provided in injectable dosage unit form, e.g. as a solution, suspension or emulsion, formulated in conjunction with the above-defined pharmaceutically acceptable excipients. The dosage and method of administration, however, depends on the individual patient to be treated. Said pharmaceutical composition, can be administered in any suitable dosage known from other peptide dosage regimens or specifically evaluated and optimized for a given individual. For example, the active agent (i.e. the fusion peptide of the present invention or the Cbl-b binding peptide) may be present in the pharmaceutical composition in an amount from 1 mg to 10 g, preferably 50 mg to 2 g, in particular 100 mg to 1 g. Usual dosages can also be determined on the basis of kg body weight of the patient, for example preferred dosages are in the range of 0.1 mg to 100 mg/kg body weight, especially 1 to 10 mg/kg body weight (per administration session). The administration may occur e.g. once daily, once every other day, once per week or once every two weeks. As the preferred mode of administration of said pharmaceutical composition, is parenteral administration, the pharmaceutical composition according to the present invention is preferably liquid or ready to be dissolved in liquid such sterile, de-ionised or distilled water or sterile isotonic phosphate-buffered saline (PBS). Preferably, 1000 µg (dry-weight) of such a composition comprises or consists of 0.1-990 µg, preferably 1-900µg, more preferably 10-200µg active agent according to the present invention, and optionally 1-500 µg, preferably 1-100 µg, more preferably 5-15 µg (buffer) salts (preferably to yield an isotonic buffer in the final volume), and optionally 0.1-999.9 µg, preferably 100-999.9 µg, more preferably 200-999 µg other excipients. Preferably, 100 mg of such a dry composition is dissolved in sterile, de-ionised/distilled water or sterile isotonic phosphate-buffered saline (PBS) to yield a final volume of 0.1-100 ml, preferably 0.5-20 ml, more preferably 1-10 ml.
Herein, the term "binding" or "binds" entails that molecule A (such as a molecule binding to Cbl-b, such as the Cbl-b binding peptide) has a dissociation constant (also called "affinity") in regard to molecule B (such as Cbl-b, specifically the TKB domain thereof) that is lower than (i.e. "stronger than") 1000 nM, preferably lower than 100 nM, more preferably lower than 50 nM, even more preferably lower than 10 nM, especially lower than 5 nM.
Preferably, the patient to be prophylactically or therapeutically treated is a mammal, especially a human. Typically, the patient (who preferably has a fungal infection as defined herein, and especially has been diagnosed with a fungal infection as defined herein) is in need of the inventive treatment. In the context of the inventive treatment for prevention of a fungal infection, the patient may be a patient that does not have the fungal infection but is at risk of developing the fungal infection, such as an immune-compromised patient.
Herein, the term "TKB binding peptide" refers to a Cbl-b binding peptide which binds to the TKB domain of Cbl-b.
"Comprising" is used as an open ended term and means that other elements may also be present. "Consisting of" is used as a closed term, wherein, accordingly the described element or group of elements of the invention consist only of the recited element(s). An embodiment of the invention that has been described as comprising a certain element or group or elements, if not stated otherwise, may also only consist of this element or group of elements.
The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".
Following embodiments further define the present invention:
SFNPYEPX1X2X3,
wherein X1-X3 are selected independently from any amino acid, preferably containing proteinogenic and/or non-proteinogenic amino acids as described above, more preferably wherein the Cbl-b binding peptide is a peptide fragment of Syk.
STVSFNPYEPX1X2X3X4WX5,
wherein X1-X5 are selected independently from any amino acid, preferably containing proteinogenic and/or non-proteinogenic amino acids as described above, more preferably wherein the Cbl-b binding peptide is a peptide fragment of Syk.
The present invention is further illustrated by the following figures and examples, without being restricted thereto.
Mice: Cbl-bΔ/Δ, Cbl-bC373A/C373A and Rag2Δ/Δ deficient mice have been described previously and were bred on a C57BI/6 background. Mouse genotypes were assessed by PCR. Of note, only age- and sex-matched littermates from respective breedings were used for experiments. All mice were bred and maintained in accordance with ethical animal license protocols complying with the current Austrian law.
Generation of bone marrow chimeric mice: To generate bone marrow chimeric mice, 8 week old wild type or Rag2Δ/Δ deficient C57BL6 mice were split dose irradiated (2 x 6 Gy) and reconstituted 18h after the second irradiation with 3 million donor cells by intravenous injection. Experiments were carried out 8 weeks later to allow for full reconstitution. Reconstitution efficiency under the applied conditions was tested by using congenic CD45.1 recipient mice and staining of splenocytes for CD45.1 (recipient derived) and CD45.2 (donor derived), 8 weeks after reconstitution, and was routinely >98%.
Isolation and differentiation of neutrophils, monocytes, and dendritic cells: Neutrophils were isolated from tibias and femurs of mice using a Percoll-gradient (GE Healthcare) as previously described (Boxio et al., 2004) or were purified using MACS technology (Miltenyi) after Ly-6G labeling. Bone marrow monocytes were purified by FACS sorting after labeling with antibodies specific for CD11b, Ly-6G, and Ly-6C. Splenic dendritic cells were FACS sorted after labeling with antibodies to CD19, TCRβ, Ly-6G, and CD11c. Purified neutrophils, monocytes, and splenic dendritic cells were cultured in RPMI-1640 medium (PAA). BM-DC or BM-M were obtained by differentiation from bone marrow precursor cells. Peripheral blood monocytes or neutrophils were obtained by sub-mandibular bleeding and collected in potassium EDTA tubes (Sarstedt, Germany). Red blood cells were lysed using an Ammonium-Chloride-Potassium (ACK) buffer.
Flow cytometry: Antibody labeling of leukocytes was carried out in FACS staining buffer (PBS supplemented with 2% FBS and 2mM EDTA) on ice for 30min after blocking of Fc-receptors. Cell viability was assessed with the Fixable Viability Dye eFluor780 (eBioscience). Immune cell recruitment was assessed by Collagenase 4 (Worthington)/DNAse (Roche) digestion of organs, viability staining, Fc-blocking, antibody staining, and subsequent flow cytometric analysis. Samples were acquired with a BD LSRII flow cytometer and data were analyzed using FlowJo software (Treestar).
Measurements of cytokine release, ROS production, Caspase-8 activity: Cytokine secretion by cell culture supernatants or in the peritoneal lavage was assessed with ELISA kits purchased from eBioscience (IL-1β, TNF, IL-6, IL-12, IL-23) or R&D Systems (IL-1Ra) according to the manufacturer's instructions. Production of reactive oxygen species (ROS) was measured with as assay using luminol as the probe in real-time over 90min as described previously (Bourgeois et al., 2009). Caspase-8 activity was assessed using the CaspGLOW assay (Promega) according to the manufacturer's instructions.
Phagocytosis assay: For phagocytosis assays Candida albicans (strain SC5314) were Alexa 488 labeled in 100mM HEPES buffer (pH 7.5). BM-DC or BM-M were co-cultured with labeled Candida albicans at 37°C for 45 minutes. Fluorescence by fungal cells adherent to, but not phagocytosed by, phagocytes, were quenched with trypan blue (Sigma Aldrich), and the rate of phagocytosis was assessed by flow cytometry.
Killing assay: To assess the fungicidal activity, the indicated immune cells were plated in replicates at a density of 1 x 105 cells/well in 96-well plates. Cells were incubated with Candida albicans at a multiplicity of infection of 1:500 for 24h. After incubation, phagocytes were fixed by the addition of paraformaldehyde at a final concentration of 4%. Subsequently, Candida albicans were stained with Crystal Violet (Sigma) and killing was assessed by a comparison of colony numbers in wells with or without phagocytes.
Mouse infection and clodronate liposome treatment: 8-10 week old mice were infected with 105 colony forming units (CFU) of C. albicans (strain SC5314), 106 colony forming units (CFU) of C. dubliniensis, or 105 CFUs of Listeria monocytogenes (strain EGD) intravenously. C. albicans CFUs were assessed from various organs as described (Wirnsberger et al., 2014). For clodronate or PBS liposome treatment experiments, mice were injected intraperitoneally with 100ul/10g mouse weight of liposomes both 24h before and 24h after intravenous C. albicans infections. Clodronate or PBS control liposomes were obtained from clodronateliposomes.org, and handled according to the manufacturer's instructions. All animal infection experiments were evaluated by the ethics committee of the Medical University of Vienna and approved by the Federal Ministry of Science and Research, Vienna, Austria.
BM-M and BM-DC stimulation experiments: BM-M and BM-DC were stimulated with C. albicans (strain SC5314), Zymosan (Invivogen), Curdlan (Invivogen), TDM (Invivogen), CpG (Invivogen), LPS (Invivogen), or C. albicans hyphae. For inhibition experiments cells were treated with the Syk inhibitor R406 (Selleckchem), anti-Dectin-1 antibody (Invivogen), MG132 (Calbiochem), or Dynasore (Sigma-Aldrich), 30min prior to stimulation (MOI: Immune cell: C. albicans = 1:2). Fungal isolates of Candida krusei, Candida glabrata, and Candida dubliniensis were kindly provided by the Allgemeines Krankenhaus Vienna (AKH). Fungal isoates were tested by PCR to verify the respective species.
Quantitative PCR: mRNA was isolated using the SV-Total RNA Isolation System (Promega) or Rneasy Kit (Qiagen), reverse transcribed using the Reverse Transcription System (Promega) or the iScript cDNA synthesis Kit (Biorad). QPCR was performed with KAPA SYBR Fast Universal (Peqlab).
Western blotting and Immunoprecipitation: Western blotting was performed according to standard protocols. Blots were blocked for 1 hour with 5% bovine serum albumin in 1 x TBS/0.1% Tween-20, or 4% BSA in 1 x TBS/0.1% Tween-20 for phosphorylated epitopes, followed by overnight incubation at 4°C with primary antibodies. Subsequently, blots were washed three times in 1 x TBS/0.1% Tween-20 for 15 min, followed by incubation with HRP-conjugated secondary antibodies (1:2500; GE Healthcare # NA9340V) for 45 min at room temperature, washed three times in 1 x TBS/0.1% Tween-20 for 15 min and visualized using enhanced chemiluminescence (ECL Plus, Pierce). Anti-β-Actin mAbs were used to control for protein loading. For Cbl-b-immunoprecipitation experiments, cells were stimulated with C. albicans, lysed in RIPA buffer (Cell Signaling Technology) containing phosphatase and proteinase inhibitors (Halt Protease and Phosphatase inhibitor Cocktail; Thermo Scientific), pre-cleared with Protein A/G Plus Agarose beads (Santa Cruz Biotechnology), and incubated overnight with an anti-Cbl-b antibody. Precipitates were analyzed by Western blotting using the indicated antibodies. For Dectin-1 and Dectin-2 immunoprecipitates, BMM of the indicated genotypes were pretreated with E-64 (10µM) for 30 min, infected with C. albicans yeast or hyphae for the indicated periods of time, lysed in RIPA buffer containing 2% SDS on ice for 30min, and diluted with RIPA buffer without SDS for a final SDS concentration of 0.4%. After brief sonication, lysates were immunoprecipitated with an anti-Dectin-1 or anti-Dectin-2 antibody at a concentration of 1µg/ml. Precipitates were analyzed by Western blotting with the indicated antibodies.
In vitro ubiquitination assays: Reaction mixtures for in vitro ubiquitination assays contained 100nm Ube1 (purified in-house), 500Nm UbcH5b (Enzo Life Sciences), 150nm full length human Cbl-b (Abnova) or human Cbl-b29-483, or a catalytically inactive human C373A Cbl-b29-483 mutant, and 100mM ubiquitin (Sigma-Aldich) with 300 nm of substrate in reaction buffer (20mM Tris-Hcl pH 7.5, 150mM NaCl, 10mM MgCl2 and 1mM DTT). Substrates were active (phosphorylated) human Syk (Merck Millipore) and inactive (non-phosphorylated) human Syk-GST (Life technologies). 1MM ATP was added to initiate the reaction and incubated at 37°C for 30 min. For TKB binding peptide mediated Cbl-b inhibition, Cbl-b was pre-incubated with the TKB binding peptide at a concentration of 100µM for 20 min at 24°C, before addition to the reaction mixture. Assays were resolved by SDS-PAGE, transferred on a nitrocellulose membrane (GE Healthcare) and blotted with an anti-ubiquitin antibody, or an anti-Syk antibody.
Production of recombinant Cbl-b29-483 and C373A Cbl-b29-483: Cbl-b29-483 and C373A Cbl-b29-483 coding sequences were cloned into the pGEX-2TK vector to obtain Gluthathione-S-transferase (GST) fusion proteins. Expression in Escherichia coli BL21 was induced with 0.3 mM isopropyl-D-thiogalactopyranoside at OD600 of 1.0 in Terrific Broth medium, and incubated overnight at 20°C. Cells were lysed in lysis buffer (20mM Tris-Hcl pH 7.5, 100mM NaCl, 1mM EDTA, 0.5% Triton X-100), sonicated briefly three times, and incubated overnight with Glutathione Sepharose 4B (GE Healthcare) at 4°C. Subsequently, Sepharose beads were washed 5 times with lysis buffer, and bound proteins were eluted with elution buffer (50mM Tris-Hcl, pH 8.0, and 20mM reduced glutathione). Eluates were tested for size and purity with SDS-PAGE and Western blotting.
Histopathology: For histopathology analyses, kidneys were fixed in 10% neutral buffered formalin, processed according to standard procedures, embedded in paraffin, and sectioned. 2 µm thick sections were stained with haematoxylin and eosin (H&E), periodic-acid-Schiff (PAS), or anti-Ly-6G (Biolegend; clone 1A8). Immunohistochemistry was performed with a primary antibody dilution of 1:100 with DAB enhancement after antigen retrieval (BOND Epitope Retrieval kit, Leica). 4 µm sections were stained with Gomori Methenamine Silver (GMS; Merck). Stained slides were scanned using a Pannoramic slide scanner (3D Histech) and evaluated for severity of inflammation and intra-lesional fungal burden based on the following criteria:
Definiens Tissue StudioTM software was applied to evaluate the extent of infiltration by Ly-6G+ cells in affected kidneys. Regions and cells of interest were manually classified in representative areas of slides to train the software algorithm to identify Ly-6G positive target objects and exclude non-target objects. Target objects were identified on the basis of morphology and positive immunohistochemical staining. The optimized algorithm was then applied to analyze all slides automatically. The validity of the analysis was confirmed by histopathologic verification of representative slides from both groups.
Peptide synthesis: Solid phase peptide synthesis (SPPS) was used for TKB binding peptide synthesis on an ABI 433a Peptide Synthesizer (Applied-Biosystems). Peptides were purified with an Ultimate 3000 RP-HPLC system (Thermo Scientific), and analyzed with a 4800 MALDI TOF/TOF analyzer (ABSciex). Lyophilized peptides were resolved in PBS and stored at -80°C.
Immunofluorescence: BM-CD were seeded on µ-slides (IBIDI) 24h before infection with C. albicans. After infection cells were fixed in 4% PFA on ice for 20min, permeabilized (0.2% Triton X-100 in PBS) for 10 min at room temperature, and blocked (3% BSA in PBS) for 1 hour at room temperature. Subsequently, cells were incubated with primary antibodies against Syk (1:100) or Shp-2 (1:100) in 3% BSA at overnight. Cells were then washed in PBS and incubated with an anti-rabbit-Alexa555 antibody (1:500) in 3% BSA at room temperature for 1h. After washing in PBS cells were counterstained with DAPI, and directly imaged with a Zeiss LSM780 Confocal microscope.
NanoLC-MS Analysis: The nano HPLC system used for this study was an UltiMate 3000 HPLC RSLC nano system (Thermo Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Scientific), equipped with a Proxeon nanospray source (Thermo Scientific). Peptides were loaded onto a trap column (Thermo Fisher Scientific, Amsterdam, Netherlands, PepMap C18, 5 mm x 300 µm ID, 5 µm particles, 100 Å pore size) at a flow rate of 25 µL min-1 using 0.1% TFA as mobile phase. After 10 min the trap column was switched in line with the analytical column (Thermo Fisher Scientific, Amsterdam, Netherlands, PepMap C18, 500 mm x 75 µm ID, 2 µm, 100 Å). Peptides were eluted using a flow rate of 230 nl min-1, and a binary 2h gradient for 165 min. The gradient was started with 98% water/formic acid (99.9/0.1; v/v) and 2% water/acetonitrile/formic acid (19.92/80/0.08; v/v/v), and increased to 35% water/formic acid (99.9/0.1; v/v) and 65% water/acetonitrile/formic acid (19.92/80/0.08; v/v/v) over 120min, followed by a gradient to 10% water/formic acid (99.9/0.1; v/v) and 90% water/acetonitrile/formic acid (19.92/80/0.08; v/v/v) over 5min, kept at this ratio for 5 min and subsequently decreased over 2 min back to 98% water/formic acid (99.9/0.1; v/v) and 2% water/acetonitrile/formic acid (19.92/80/0.08; v/v/v) for equilibration at 30°C. The Q Exactive mass spectrometer was operated in data-dependent mode, using a full scan (m/z range 370-1650, nominal resolution of 70 000, target value 3E6) followed by MS/MS scans of the 12 most abundant ions. MS/MS spectra were acquired using normalized collision energy 27%, isolation width of 2 and the target value 1E5. Precursor ions selected for fragmentation (charge state 2 and higher) were put on a dynamic exclusion list for 10 sec. The underfill ratio was set to 20% resulting in an intensity threshold of 4E4. The peptide match feature and the exclude isotopes feature were enabled.
Mass spectrometry data analysis: For peptide identification the RAW-files were loaded into Proteome Discoverer (version 1.4.0.288, Thermo Scientific). All created MS/MS spectra were searched using the search engine node MSAmanda (Dorfer et al., 2014) against the flybase sequence database (22,256 sequences; 20,222,850 residues) and also against the Swissprot sequence database, using the taxonomy human (20,170 sequences, 11,318,213 residues). The following search parameters were used: Beta-methylthiolation on cysteine was set as a fixed modification, oxidation on methionine, acetylation on lysine, phosphorylation on serine, threonine and tyrosine, deamidation on asparagine and glutamine, and ubiquitinylation on lysine were set as variable modifications. Monoisotopic masses were searched within unrestricted protein masses for tryptic peptides. The peptide mass tolerance was set to ±5 ppm and the fragment mass tolerance to ± 0.03 Da. The maximal number of missed cleavages was set to 2. The result was filtered to 1% FDR using Percolator algorithm integrated in Proteome Discoverer. The localization of the sites of variable modifications within the peptides was performed with the tool ptmRS, integrated in Proteome Discoverer and based on phosphoRS (Taus et al., 2011).
Data Analysis and Statistics. All values in the paper are given as means ± standard deviation unless stated otherwise. All in vitro experiments were reproduced 3-5 independent times. In vivo experiments were reproduced 2-6 times. Figures and statistical analyses were generated using PraphPad Prism software (GraphPad Software). No mice were excluded from the analyses and histopathologic analyses using Definies Tissue studio software were performed blindly. Mice were allocated to experimental groups based upon their genotypes and randomized within their sex- and age matched groups. Since mice were inbred and sex- and age-matched, similar variance between experimental cohorts was assumed. An a priori sample size estimation was not performed. Group sizes were based upon the empirically assessed variability of the model systems or assays used, and groups contained as many mice as possible to minimize type I and type II errors. Data were analyzed using the unpaired two-tailed Student's t-test, or two way ANOVA, as indicated. For survival analyses, log rank tests were performed. P values < 0.05 were accepted as statistically significant.
The following antibodies were used in this study:
The following PCR primers were used in this study:
To test for a potential role of Cbl-b in anti-fungal immunity, Cbl-bΔ/Δ and control Cbl-b+/+ littermates were systemically challenged with a lethal dose of Candida albicans. In addition, Cbl-bc373A/c373A mice, carrying catalytically inactive Cbl-b (Paolino et al., 2011), were infected with Candida albicans. Loss of Cbl-b protein (
To gain insight into the cellular basis of the observed protective effect, bone marrow chimeric animals were generated by lethally irradiating C57Bl/6 recipient mice and reconstituting them with bone marrow from syngeneic Cbl-bΔ/Δ, Cbl-bC373A/C373A, or Cbl-b+/+ littermate mice. After reconstitution, the cohorts were infected with a lethal dose of Candida albicans. Loss of Cbl-b in the radiosensitive hematopoietic compartment mirrored full body Cbl-b deficiency (
To test for potential effects of Cbl-b deficiency on the development or maintenance of innate immune cells relevant for the anti-fungal defense, cellularity and cellular composition of the main innate immune lineages in the bone marrow and spleen of Cbl-bΔ/Δ and Cbl-b+l+ littermates were assessed. Slightly elevated levels of both bone marrow and splenic Ly-6G+ neutrophils and splenic NK cells could be observed (
To test whether the protective effect of Cbl-b deficiency could be extended to other routes of infection, a model of cutaneous Candida albicans infection was used (Igyarto et al., 2011). In this model an increased resistance of Cbl-bΔ/Δ mice to skin colonization by Candida albicans was again observed, when compared to littermate controls, as evidenced by a significantly decreased fungal burden (
Fungicidal activity within the innate immune system is mainly attributed to neutrophils, macrophages and DC (Drummond et al., 2015). To directly investigate the effect of Cbl-b deficiency on the anti-fungal activity of these cell types, in vitro killing assays were performed by co-culturing bone marrow neutrophils from uninfected mice, bone marrow-derived macrophages (BM-M), bone marrow-derived dendritic cells (BM-DC), bone marrow monocytes, and splenic DC, isolated from the bone marrow or the spleen, respectively, of uninfected mice, with Candida albicans. No significant difference in fungicidal activity could be observed, when comparing Cbl-bΔ/Δ, Cbl-bC373A/C373A, and Cbl-b+/+ bone marrow neutrophils (
In line with previous observations, pharmacologic inhibition of phagocytosis using Dynasore abrogated BM-DC mediated killing. Likewise, antibody mediated blocking of Dectin-1 significantly reduced Candida albicans killing and reduced the increased fungal killing in both Cbl-bΔ/Δ and Cbl-bC373A/C373A BM-DC to levels observed in Cbl-b+/+ cells (
Candida albicans mediated activation of BM-DC also triggers Syk dependent activation of the inflammasome, that enables cleavage of pro-interleukin-1β (IL-1β) by Caspase-8 (Gringhuis et al., 2012; Zwolanek et al., 2014). Significantly increased activation of Caspase-8 (
To monitor the transcriptional response to Candida albicans infection, mRNA levels of pro-inflammatory cytokines were assessed. Significantly increased transcript levels of IL-1β, IL-6, TNF, IL-23, and IL-12 in Cbl-bΔ/Δ and Cbl-bC373A/C373A BM-DC when compared to Cbl-b+/+ control cells were observed (
To further test for the contribution of Dectin-1, cytokine secretion by BM-DC of all three genotypes in response to increasing doses of zymosan, a Candida albicans cell wall homogenate, or curdlan, a purified β-glucan was compared. Stimulation with zymosan and the Dectin-1 agonist curdlan resulted in an increased release of IL-1β, IL-6, and TNF by Cbl-bΔ/Δ and Cbl-bC373A/C373A BM-DC (
The data so far showed that Cbl-b deficiency or a lack of Cbl-b activity leads to a severe de-regulation of the anti-fungal immune response in vitro. To address, whether the observed differences could be recapitulated in immune cells recruited to inflammatory sites, Cbl-bΔ/Δ and Cbl-b+/+ mice were challenged by intraperitoneal infection with Candida albicans and their anti-fungal activity was assessed 24h later. Loss of Cbl-b led to an enhanced ROS response by total infiltrating immune cells into the peritoneal cavity. Increased ROS production was already observed in Cbl-bΔ/Δ, when compared to Cbl-b+/+, infiltrating cells without further re-stimulation with Candida albicans (
The data indicated that the protective effect of Cbl-b deficiency to fungal challenge was mediated by tissue resident or recruited phagocytic immune cells, but not neutrophils. To test for neutrophil intrinsic effects phagocytic immune cells were depleted in vivo using clodronate liposomes. Clodronate liposomes have been reported to deplete macrophages, subpopulations of monocytes, and subsets of DC, while leaving neutrophils unaffected (Buiting et al., 1994; Buiting et al., 1996; Qian et al., 1994; Sunderkotter et al., 2004; van Rooijen et al., 1992). Phagocytes were depleted 24h before, and 24h after systemic infection with Candida albicans and both weight loss and survival were monitored over time. Depletion of phagocytes led to a significant increase in weight loss in both Cbl-b+/+ and Cbl-bΔ/Δ mice compared to the control PBS liposome treated cohorts (Qian et al., 1994) (
To assess whether loss of Cbl-b or Cbl-b ligase function altered the magnitude of the signaling response mounted by BM-DC, tyrosine phosphorylation was analyzed 30, 60, and 90 minutes after stimulation with Candida albicans. A strong increase in tyrosine phosphorylation in response to Candida albicans stimulation was observed in lysates prepared from Cbl-bΔ/Δ and Cbl-bC373A/C373A BM-DC, when compared to control Cbl-b+/+ BM-DC for all stimulation time points tested (
To test whether the E3 ligase Cbl-b could poly-ubiquitinate molecules critical for proximal CLR signaling, ubiquitination assays with recombinant proteins were performed. No poly-ubiquitination of recombinant Shp-2 was detected in these assays, but observed robust poly-ubiquitination of recombinant active Syk was observed (
To test for potential additional effects of Cbl-b deletion on proximal signaling initiated by CLR stimulation, the question was raised whether the CLR family members Dectin-1, and -2 might also serve as targets for Cbl-b mediated degradation. Thus, BM-M were stimulated with Candida albicans or Candida albicans hyphae, and tested for Dectin-1, and -2 protein levels, respectively, at various time points after stimulation. While Dectin-1, and -2 levels decreased over time in control cells, their expression levels remained constant in the absence of Cbl-b (
Based on the genetic and biochemical data it was reasoned that blocking Cbl-b-TKB mediated substrate recognition might deregulate the activation of Syk downstream of CLR stimulation. To test this hypothesis, a fusion peptide consisting of a cell penetrating peptide derived from the Drosophila Antennapedia homeodomain was designed (May et al., 2000) and a 16aa long phosphorylated peptide derived from Syk that encompasses Tyrosine 317 (Y317), reported to be required for Cbl-family-ligase mediated targeting of Syk (Sohn et al., 2003; Yankee et al., 1999; Zou et al., 2009), and the surrounding flanking regions (
Due to the observed in vitro efficacy of the inhibitory peptide it was investigated whether inhibition of Cbl family ligases might represent a novel strategy to increase immune reactivity and function towards fungal sepsis in vivo. Thus, Cbl-bΔ/Δ and Cbl-b+/+ littermate control mice were infected with a lethal dose of Candida albicans and treated two times with a high dose of the inhibitory Cbl-b TKB-binding peptide (100µg, i.p., day 0 and day 2), or left untreated. Strikingly, treating mice only twice had a significant protective effect on wild type Cbl-b+l+ mice, as evidenced by reduced weight loss and delayed mortality, when compared to the untreated control cohort (
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