Patent Application
20240263161
The present invention relates to mucin-binding targeting agents comprising peptides derived from microbial proteins that have selective binding properties for densely glycosylated mucins. Furthermore, the invention relates to use of such targeting agents to bind mucin layers covering mucosal surfaces such as gastric and colonic mucous and mucosal lining epithelia. The provided mucin-binding agents are useful for detecting and binding mucins and mucosal surfaces for biomarker and therapeutic purposes. The invention also relates to methods for the use of mucin-binding agents for targeting and delivery of therapeutic agents to mucosal surfaces.
Mucins are a large family of heavily glycosylated proteins that line all mucosal surfaces and represent the major macromolecules in body fluids1. Mucins clear, contain, feed, direct, and continuously replenish our microbiomes, limiting unwanted co-habitation and repressing harmful pathogenic microorganisms2. Mucins in the gut constitute the primary barrier as well as the ecological niche for the microbiome. Dynamic replenishment of mucin layers provides constant selection of the resident microbiome through adhesive interactions, and degradation of mucin O-glycans by members of the microbiota supply nutrients3,4,2,5. Mucin O-glycans present the essential binding opportunities and informational cues for microorganisms via adhesins, however, our understanding of these features is essentially limited to results from studies with simple oligosaccharides without the protein context of mucins and the higher order features presented by dense O-glycan motifs. Mucins are notoriously difficult to isolate due to their size and heterogeneity, and production by recombinant expression in cell lines is impeded due to difficulties with the assembly of full coding expression constructs often resulting in heterogeneous products6.There are at least 18 distinct mucin genes encoding membrane or secreted mucins in the human genome7. The large gel-forming secreted mucins may form oligomeric networks or extended bundles through inter- and intramolecular disulfide bridges in the C- and N-terminal cysteine-rich regions1. A common characteristic of all mucins is that the major part of their extracellular region is comprised of variable number of imperfect tandem repeated (TR) sequences that carry dense O-glycans, with the notable exception of MUC16 that contains a large, densely O-glycosylated N-terminal region without TRs8,9. These TR regions appear poorly conserved throughout evolution in contrast to the flanking regions of the large mucins10, and this is generally interpreted to reflect that the TR regions simply need to carry dense O-glycans without specific patterns or functional consequences.
Mucins line all mucosal surfaces and represent one of the most abundant components of body fluids including saliva5,11. Gel or polymer-forming secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6) cover the gastrointestinal tract. In the stomach a net-like layer of MUC5AC polymers forms a diffusion barrier protecting the surface lining epithelium12. In the small intestine a non-attached mucus layer exists, mainly comprised of MUC2. The large intestine, however, is covered by a thick two-layered MUC2 network with a dense inner layer that serves as a molecular sieve impenetrable by e.g. bacteria, and an outer loose MUC2 network that contains the microbiome at a distance from the surface epithelium. These mucin layers cover and protect the lining mucosa and present an obstacle for delivery of therapeutics through mucosal surfaces13.
Mucins arguably represent a last frontier in analytics of glycoproteins. Most mucins are extremely large and heterogenous glycoproteins that are resistant to conventional glycoproteomics strategies that are dependent on proteolytic fragmentation and sequencing14-16, and despite increasing knowledge of O-glycosites9, identification of actual sites of glycosylation in mucins is essentially limited to MUC117,18,19, lubricin20, and the large N-terminal mucin-like region of MUC169. Current understanding of mucins, their glycosylation, and their functions is therefore still highly limited.
Secreted protease of C1 esterase inhibitor (StcE) from Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zinc metalloprotease with remarkable ability for cleaving densely O-glycosylated mucins and mucin-like glycoproteins. StcE is thought to serve in colonic mucin degradation facilitating EHEC adherence to the epithelium and subsequent infection21,22. Recent studies demonstrated that StcE has selective substrate specificity for S/T-X-S/T motifs with a requirement for O-glycans at the first S/T residue23. The StcE glycoprotease was demonstrated to cleave a wide array of isolated mucins or cell-membrane mucins endogenously expressed by cancer cell lines, including cell surface mucins, such as MUC1 and MUC16, and mucin-like O-glycoproteins such as CD43 and CD45, revealing its broad substrate specificity with mucins and mucin-like glycoproteins23.
Moreover, a catalytically inactive mutant of StcE (StcEE447D) has glycan-binding properties for the dST core1 O-glycan as evaluated by printed glycan arrays24, but also exhibit general binding properties for mucins23,25-27. It has been proposed that StcE plays a role in adherence of EHEC to the intestinal epithelium by binding to mucins24,25,28. More recently, Bertozzi and colleagues took advantage of a classical strategy to employ the catalytic unit of an enzyme for use as a binding molecule29. This involves inactivating the catalytic function of the enzyme and retaining the substrate binding properties of the catalytic unit of the enzyme. Thus, the catalytically inactive mutant of StcE (StcEE447D) was used for binding studies with tissue sections and binding to mucin secreting cells by use of histological tissue sections was demonstrated27 [PCT/US2019/060346]. StcE has also been suggested to bind O-glycans on a glycan array and in particular to the core1 disialyl-T (dST) O-glycan27. Moreover, a recent report described use of the StcEE447D mutant for affinity isolation of O-glycoproteins demonstrating that the enzyme can be used to enrich mucins and a variety of other O-glycoproteins many of which are not mucins or mucin-like such as LRP830.
State-of-the-art technologies to capture the informational content of mucins are confined to studies with synthetic and isolated O-glycans31, synthetic and chemoenzymatically produced short glycopeptides32, and synthetic glycopeptides as well as non-natural polymers33,34; all of which are rare commodities that do not reflect the complex information captured in distinct human mucins by their display of patterns and structures of O-glycans. With the advent of the facile nuclease-based gene engineering technologies it has become possible to engineer mammalian cells with combinatorial knockout (KO) and knockin (KI) of glycosylation genes to display subsets and distinct features of the glycome on the cell surface or on secreted reporter proteins in order to probe biological interactions dependent on glycans35-37 [US patent applications US20210087587 and US20190330601]. The genetic engineering provides opportunities for interpretation and dissection of the glycosylation genes, biosynthetic pathways, and structural features required for identifiable interactions with the cell library38. The cell-based glycan array is a sustainable platform to display the human glycome and enabling wide surveying of the informational content of human glycans. Use of the cell-based glycan display is fundamentally different from printed glycan arrays in that the primary read-out is consequences of loss/gain of glycosyltransferase genes that are used to predict structural glycan features. Printed glycan arrays provide direct information of glycan haptens involved in interactions, while the cell-based array provides comprehensive knowledge of genes regulating the expression of glycan features.
The cell-based glycan array strategy was previously used to express designed protein reporter constructs containing the high density O-glycan regions (O-glycodomains) derived from the stem region of GP1bα and TRs from several different mucins36. The reporters were transiently expressed in HEK293WT cells and used to demonstrate that bacterial adhesins from Streptococcus show differential binding to cells displaying different mucin reporters. These studies suggested that the adhesins recognize specific O-glycan structures in ways influenced by their presentation on the protein or mucin backbone. Analysis of the sequences used for the reporter constructs derived from the mucin TRs and GP1bα did not reveal simple common sequence motifs shared among those providing binding for the Siglec-like adhesins, and thus, the data could not be used to define the recognition motifs in further detail. Recognition of “clustered saccharide patches” orchestrated by positions, spacing and direct interactions of multiple glycans in a protein was earlier proposed to provide expanded binding specificities and high affinity interactions, and evidence in support of this has been found with all types of glycoconjugates39,40.
Printed glycan arrays have transformed the field of glycosciences and served as essential tools for exploring the interactome of glycans and proteins41; however, studies have also resulted in the emergence of an interesting conundrum in explaining diversity of pathogen interactions and their host tropism in nature39,42. Results from printed glycan arrays indicate that relatively few distinct glycan motifs serve as common ligands for many microbial adhesins and glycan-binding proteins42. The core structural motifs recognized, typically only 3-5 monosaccharides, are even more limited since glycans are built on common scaffolds with units such as N-Acetyllactosamine (LacNAc)42,43 Many host-pathogen interactions identified involve terminal sialic acid residues on common structural motifs, and while the glycosidic linkages, the underlying core structures, and numerous modifications of sialic acids44, do vary to a degree, the overall structural permutations are still somewhat limited.
Current knowledge of the molecular basis for host-pathogen interactions are therefore incomplete, and essentially limited to binding and recognition of different simple oligosaccharide structures by lectins, adhesins, agglutinins and other carbohydrate-binding modules42,43. Such oligosaccharides may be widely found on glycolipids, glycoproteins including mucins, proteoglycans, other types of glycoconjugates, and as free oligosaccharides found widely on cells and in body fluids throughout the body45. Binding to such glycan epitopes by microbial glycan-binding molecules therefore have limited cell-type and organ specificity.
The present invention relates to microbial peptide modules, that bind to select human and/or other mammalian mucins and do not bind to simple oligosaccharides. Microbial peptide modules of the invention bind to the tandem repeat regions of human mucins when these have clusters of O-glycans attached, as found on mucins in normal and diseased mucosa. The present invention also relates to methods of use of such microbial peptide modules in mucin-binding targeting agents for binding to mucins found in the mucous layers of human lining epithelia. Moreover, the present invention provides the use of such binding modules to detect mucins and mucous layers, and use of these modules to deliver pharmacological agents to mucosal surfaces.
The mucin-binding targeting agents present several advantages, including high selectivity for preferred mucins, such as MUC5Ac, and preference for binding non-truncated sugars over truncated sugars. Moreover, the mucin-binding targeting agents bind selected mucins with high affinity, such as in the nanomolar range.
The nanomolar range binding affinities of the mucin-binding targeting agents are improved markedly compared to other lectin-based systems (micromolar) and on par with binding affinities achieved by antibodies. Moreover, the high selectivity ensures that any attached payload can be delivered with high precision to the intended tissue expressing the targeted mucin(s), while the preference for non-truncated sugars limits off-target binding to truncated sugars, e.g. STn and Tn, which are associated with shed degraded mucins from the mucus.
Average MFI±SEM values from three independent experiments are shown. Panel e Flow cytometry analysis of X409-GFP binding (1 μg/ml) to MUC2#1, MUC5AC, MUC7 and MUC1 reporters expressed on glycoengineered HEK293 cells (core2, diST, mSTa, T, Tn, core3, and STn). Structures of glycans are shown with symbols drawn according to the Symbol Nomenclature for Glycans (SNFG) format44. Note that colors are omitted but illustrated HexNAc (open/grey), Hex, and sialic acid represent GalNAc/GlcNAc, Gal and NeuAc.
It is an object of the present invention to provide novel mucin-binding peptides that exhibit select binding properties for mucins and O-glycodomains in glycoproteins and to provide mucin-binding targeting agents comprising such peptides.
It is an object of the present invention to provide novel mucin-binding peptides that exhibit select binding properties for mucins and mucin tandem repeat regions, which binding is not dependent on the structures of O-glycans attached to these.
It is an object of the present invention to provide novel mucin-binding peptides that exhibit select binding properties for mucins and mucin tandem repeat regions which binding is independent on the structures of the O-glycans attached to these mucins.
An object of the present invention relates to the mucin-binding properties of the X409 peptide module and related sequences and use of these to bind mucins for diagnosis of disease.
An object of the present invention relates to the mucin-binding properties of the X409 peptide module and related sequences and use of these to bind mucins for therapeutic purposes.
An object of the present invention relates to the mucin-binding properties of the X409 peptide module and related sequences and use of these for delivery of pharmacological agents to mucosal surfaces.
An object of the present invention relates to methods of using the X409 peptide module and other peptides and peptide modules to obtain mucin-binding properties for pharmaceutical formulations.
The present invention relates to a peptide or peptide module (designated X409) found in the Secreted Protease of C1 Esterase Inhibitor (StcE) bacterial protease and other bacterial proteins and peptide modules that have select mucin-binding properties.
It is an object of the present invention to provide related X409 binding modules with high sequence similarity within bacteria.
It is an object of the present invention to provide related X409 binding modules with lower sequence similarity within bacteria, such as 65% sequence identity or more, e.g. such as such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more thereto.
The present invention provides solutions to the objects above.
The present invention provides the unique binding specificity of a small peptide module X409 with highly select binding to mucins with clusters of O-glycans attached.
The present invention provides the unique binding specificity of the small peptide module X409 with binding to mucins with clusters of O-glycans and with binding to such mucins with different types of O-glycan structures attached.
The present invention provides multiple X409-related mucin-binding peptide and peptide modules, and mucin-binding peptides and peptide modules that are not related to X409.
The present invention provides small peptide sequence modules with binding to select mucin tandem repeat regions and containing clusters of different types of O-glycans.
It is contemplated that mucin-binding targeting agents are for use in a mammal such as e.g. a human.
The mucin-binding targeting agents may be used as a medicament in a human or on an animal, such as in veterinary care or animal health.
The mucin-binding modules or polypeptides of the invention bind to densely O-glycosylated mucins and mucin-like glycoproteins, i.e. to clusters of O-glycans with 2,3,4,5,6,7, or 8 consecutive O-glycans attached to adjacent Ser/Thr residues often arranged in multiple consecutive patterns.
The term “isolated” as used herein in relation to peptides refers to amino acid sequences which have been taken out of their native environment. Thus, an isolated peptide is a non-native peptide which may be a part (or sub-sequence) of a larger peptide or protein. Isolated peptides are identified and selected based on their affinity for preferred mucins and used in mucin-binding targeting agents, which can be chimeric construct that may comprise also a payload. The chimeric constructs forming the mucin-binding targeting agents may be produced by recombinant expression in a host cell, such as a bacteria.
The term “binding affinity” is used herein to describe the strength of interaction between to binding partners, such as the mucin-binding targeting agent (or the isolated peptide) and a mucin, such as MUC5AC or MUC1. The binding affinity may be quantified by determination of the dissociation constant of said interaction. A low dissociation constant indicates a strong interaction (or binding).
The term payload as used herein refers to a moiety that is intended to be delivered to a tissue by the binding of a mucin-binding targeting agent as provided herein. The payload is thus attached to said mucin-binding targeting agent by a binding moiety.
The binding moiety may be e.g. a peptide linker, an ester, a lipid anchor, avidin, streptavidin, biotin, or another binding moiety such as an antibody, or a nanobody.
The binding moiety may be able to undergo in vivo acid hydrolysis or may comprise a protease site that can undergo cleavage to ensure the mucin-binding-domain is not delivered e.g. with a bioactive peptide to be taken up systematically.
The term payload may refer to an entire complex, such as a nanoparticle, liposome, vesicle, which contains a bioactive compound to be delivered or it may refer to a bioactive compound, stain, or e.g. a detectable marker. It is contemplated that when the payload is a liposome or another vesicle it may be attached to the mucin-binding targeting agent by a binding moiety in the form of a lipid anchor, or by a different moiety inserted into the liposome or vesicle. In the latter case the moiety is then bound or attached to mucin-binding targeting agent via the binding moiety.
When the payload is a liposome a bioactive peptide or protein, oligonucleotide, or other therapeutic agents such as a therapeutic peptide may be inside the liposome.
Therapeutic peptides may also be attached to the binding moiety, or may form chimeric proteins with the mucin-binding targeting agent, in which the binding moiety may be a peptide bond or a peptide linker.
Therapeutic peptides and proteins for use in or as payload may be selected from the group comprising:
Neuroendocrine protein 7B2, Acyl-CoA-binding domain-containing protein, Adrenomedullin, Proadrenomedullin NApelin-13 , Apelin, Gastrin-releasing peptide, Neuromedin-C, Neuromedin-B, Bradykinin, T-kinin, Calcitonin, Katacalcin, Calcitonin gene-related peptide 1, Calcitonin gene-related peptide 2, Islet amyloid polypeptide, CART Cocaine- and amphetamine-regulated, Cerebellin -4, Cerebellin-1, Cerebellin-2, Cerebellin-3, AL-11, Chromogranin-A, EA-92, ER-37, ES-43, GR-44, GV-19, LF-19, Pancreastatin, SS-18, Vasostatin, WA-8, WE-14, CCB peptide, GAWK peptide, Secretogranin, Secretoneurin, Kininogen, Big endothelin-1, Endothelin, Neuropeptide AF, Neuropeptide FF, Neuropeptide SF, Neuropeptide NPSF, Neuropeptide NPVF, Neuropeptide RFRP Prolactin-releasing peptide, Galanin, Galanin message-associated peptide, Galanin-like peptide, Cholecystokinin, Big gastrin, Gastrin, Gastric inhibitory polypeptide, Glicentin, Glicentin-related polypeptide, Glucagon, Glucagon-like peptide 1, Glucagon-like peptide 2, Oxyntomodulin, PACAP-related peptide, Pituitary adenylate cyclase-activating peptide 27, Pituitary adenylate cyclase-activating peptide 38, Secretin, Somatoliberin, Intestinal peptide PHM-27, Intestinal peptide PHV-42, Vasoactive intestinal peptide, GnRH-associated peptide 1, Gonadoliberin-1, Progonadoliberin-1, GnRH-associated peptide 2, Gonadoliberin-2, Progonadoliberin-2, Insulin-like growth factor I, Insulin-like growth factor Il, Preptin, Insulin A chain, Insulin B chain, Relaxin A chain, Relaxin B chain , Relaxin A chain, Relaxin B chain, Relaxin-3 A chain, Relaxin-3 B chain , Kisspeptin-10, Kisspeptin-13, Kisspeptin-14, Metastasis-suppressor KiSS-1, Metastin, Leptin, Melanin-concentrating hormone, Neuropeptide-glutamic acid-isoleucine, Neuropeptide-glycine-glutamic acid, Pro-MCH, Ghrelins, Obestatin, Motilin, Motilin-associated peptide, Promotilin, Adiponectin, Ubiquitin-like protein 5, Agouti-related protein, Nicotinamide phosphoribosyltransferase, Atrial natriuretic peptide ANP, Cardiodilatin-related peptide, Brain natriuretic peptide BNP , Cardiac natriuretic peptide CNP, Neurexophilin-1, Neurexophilin-2, Neurexophilin-3, Neurexophilin-4, Neuromedin-S, Neuromedin-U-25, Neuropeptide B-23, Neuropeptide B-29, Neuropeptide W-23, Neuropeptide W-30, Neuropeptide S, Large neuromedin N, Neuromedin N, Neurotensin, Tail peptide, C-flanking peptide of NPY, Neuropeptide Y, Pancreatic hormone, Pancreatic icosapeptide, Peptide YY, Nucleobindin-1, Nesfatin-1, Nucleobindin-2, Deltorphin I, Gamma-Lipotropin, γ-melanocyte-stimulating hormone, Alpha-neoendorphin, Beta-neoendorphin, Big dynorphin, Dynorphin A, Leu-enkephalin, Leumorphin, Rimorphin, Met-enkephalin, PENK, Synenkephalin, Neuropeptide 1, Neuropeptide 2, Nociceptin, Orexin-A, Orexin-B, Tuberoinfundibular peptide, Osteostatin, Parathyroid hormone-related protein, PTHrP, Beta-endorphin, Corticotropin, Corticotropin-like intermediary peptide, Lipotropin beta, Lipotropin gamma, Melanotropin alpha, Melanotropin beta, Melanotropin gamma, NPP, ProSAAS, Big LEN, Big PEN-LEN, Big SAAS KEP, Little LEN, Little SAAS, PEN, Resistin, Resistin-like beta, QRF-amide, Corticoliberin, Urocortin, Urocortin-2, Urocortin-3, Corticosteroid-binding globulin, Serpin, Angiotensin, Angiotensinogen, Cortistatin, Somatostatin, Prolactin, Substance P, C-terminal-flanking peptide, Neurokinin A, Neuropeptide K, Substance P, Neurokinin-B, Pro-thyrotropin-releasing hormone, Thyrotropin-releasing hormone, Urotensin, Neurophysin 1, Oxytocin, Arg-vasopressin, Copeptin, Neurophysin 2, Antimicrobial peptide VGF, Neuroendocrine regulatory peptide-1, Neuroendocrine regulatory peptide-2, Neurosecretory protein VGF.
The payload may be in the form of a peptide or protein or a part of a protein. The mucin-binding targeting agent and the payload form a chimeric protein. In such cases the binding moiety will be understood to be e.g. a peptide bond. Such protein or peptide payloads may be therapeutic peptides. It is also contemplated that the payload may be a receptor, a toxin, or a lectin-binding protein.
The payload may also be an enzyme, in which case the mucin-binding targeting agent may facilitate retention of the enzyme at the site of action, e.g. in the pancreas or the gut. This mode of action may be used for improving efficacy of the enzymes. Thus, enzymes include, but are not limited to, therapeutic and/or digestive enzymes. In particular, digestive enzymes targeted to the pancreas may be utilized for treatment of patients having their pancreas surgically removed. Enzymes targeted to the gut can improve feed digestion and nutrient uptake as the mucin-binding targeting agent ensures prolonged retention in the gut via specific binding to site-specific mucins carrying non-truncated sugars. The payload is not limited to any particular enzyme but may be any type of enzyme, including, but not limited to, proteases, lipases, phytases, amylase, xylanases, β-Glucanases, α-Galactosidases, mannanases, cellulases, hemicellulases, and pectinases.
It is to be understood that the mucin-binding targeting agent may be part of a fusion protein. Such fusion proteins may comprise a therapeutic agent, such as a drug, a therapeutic protein or a bioactive peptide. Fusion proteins protein can in this manner be utilized as delivery vehicles with enhanced retention at the targeted tissue, such as the nasal tissue, the pancreas or the gut.
The payload can also be a vaccine. By combining a vaccine with the mucin-binding targeting agent, the vaccine may be efficiently delivered to the mucosa. It is contemplated that delivery to the mucosa will improve the immunological protection provided by the vaccine. Without being bound by theory, herein is suggested that immunological protection can be enhanced by presentation through the mucosa to stimulate the mucosal IgA immunity.
Thus, an embodiment of the present invention relates to the mucin-binding targeting agent, wherein the payload is a vaccine, such as a viral vaccine. Accordingly, the payload may comprise one or more antigens. According, an embodiment of the present invention relates to the mucin-binding targeting agent, wherein the payload comprises one or more antigens selected from the group consisting of proteins, peptides, polypeptides or nucleic acids. In particular, the nucleic acids may be DNA or RNA, and analogues thereof. The proteins may in some variants of the payload be a glycoprotein or a polysaccharide.
A further embodiment of the present invention relates to the mucin-binding targeting agent, wherein the one or more antigens are virus-specific antigens. Yet another embodiment of the present invention relates to the mucin-binding targeting agent, wherein the virus-specific antigen originates from a virus selected from the group consisting of SARS-COV-2 virus, SARS-COV-1 virus, Corona virus, Adenovirus, Norovirus, Papillomavirus, Polyomavirus, Herpes simplex virus (HSV), Alpha herpesvirinae human herpesvirus 1, 2, 3, Human gamma herpesvirus 4, 8 (Kaposi sarcoma), Betaherpesvirinae 5, 6, 7, Varicella zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Picornavirus, Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus, Aphthovirus, Coxsackie virus, Echovirus, Paramyxovirus, Measles virus, Parainfluenza virus, Mumps virus, Respiratory syncytial virus (RSV), Metapneumovirus, Nipah virus, Hendra viruses, Orthomyxoviruses, Influenza virus, Rhabdovirus, Filovirus, Marburg virus, Ebola virus, Bornavirus, Rabies virus, Reovirus, Rotavirus, Coltivirus, Orbivirus, Norwalk virus, Calicivirus, Rubella virus, Togavirus, Flavivirus, Arbovirus, Bunyavirus, Arena virus, Poxvirus, Parvovirus, Retrovirus, Human immunodeficiency virus (HIV), Human-T cell leukemia virus, and Hepatitis A, B, C, D, G, and E viruses.
A still further embodiment of the present invention relates to the mucin-binding targeting agent, wherein the one or more antigens originates from SARS-COV-2 virus.
It is contemplated that the mucin-binding targeting agent or part thereof may be expressed recombinantly. Thus, fusion proteins comprising the mucin-binding targeting agent may be expressed recombinantly.
It is contemplated that the mucin-binding targeting agents of the invention are catalytically inactive against mucins, i.e. they lack glycomucinase activity.
The payload may also be a radionuclide or a radiopeptide. The payload may be a therapeutic agent for use in the treatment of a cancer. It is contemplated that cancers affect the expression of mucins and therefore compositions according to the present invention may be used to deliver therapeutic agents to a cancer tissue with abnormal mucin expression,
The payload may also be a stain or a detectable marker, such as a chromophore, fluorophore, or radionuclide, or other detectable markers. Examples of detectable markers include nanodots and nanoparticles such as colloidal gold, and fluorescent proteins such as GFP. Such payloads enable the use of mucin-binding targeting agents according to the present invention for use in vitro or in vivo for immunological, histological, and/or diagnostic purposes. It is contemplated that such use can be for detecting normal and abnormal mucin-expressing tissue and may therefore be used e.g. to discern healthy and/or diseased tissue, such as in cancers and/or neoplasia of mucin expressing tissue, e.g. in the colon, stomach, pancreas, mammary, fallopian tube or other epithelial tissue. It is contemplated that such use is also for cancers and/or neoplasia epithelial and non-epithelial tissue where mucin-expression is abnormal, e.g. where mucin expression is absent or low in the non-diseased state but where mucin is expressed in the diseased state.
Accordingly, an embodiment of the present invention relates to the mucin-binding targeting agent as described herein, wherein the payload is selected from the group consisting of a therapeutic agent, an enzyme, a vaccine, a peptide hormone, a small molecule drug, a detectable marker, nanoparticle, liposome, vesicle and a stain.
The mucin binding targeting agent may be used as a medicament, and may be comprised in a composition. Such a composition may further comprise a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable carrier. The compositions may be combined with excipients or coatings to form drug delivery formulations. Drug delivery formulations may be in a form of suspensions, tablets, capsules, gels, suppositories. The formulations may be oral suspensions to induce a rapid effect in combination with prolonged release. The formulation may be packaged in an excipient such as an enteric coating or a shell. The formulation may also include other mucoadhesive materials to enhance retention within the gastrointestinal tract. Compositions according to the present invention may be for oral, rectal, vaginal, buccal, ocular, nasal, or inhalation administration.
The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral, and parenteral (e.g., subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.
Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be “acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
In preferred embodiments, the mucin-binding targeting agent is administered to a subject or patient at a clinically relevant dose.
In yet other embodiments, suitable routes of administration are considered to be enteral administration, topical administration, and parenteral administration.
By enteral administration, we include methods including but not limited to oral administration, rectal administration, sublingual administration, sublabial administration, and buccal administration. Forms suitable for such administration include but are not limited to pills, tablets, osmotic controlled release capsules, solutions, softgels, suspensions, emulsions, syrups, elixirs, tinctures, hydrogels, ointments, suppositories, enemas, murphy drip, and nutrient enemas.
By topical administration, we include methods including but not limited to transdermal administration, vaginal administration, ocular administration, and nasal administration. Forms suitable for such administration include but are not limited to aerosols, creams, foams, gels, lotions, ointments, pastes, powders, shake lotions, solids (e.g., suppositories), sponges, tapes, tinctures, topical solutions, drops, rinses, sprays, transdermal patches, and vapors.
By parenteral administration, we include methods including but not limited to injection, insertion of an indwelling catheter, transdermal, and transmucosal administration. Such administration routes include but are not limited to epidural administration, intracerebral administration, intracerebroventricular administration, epicutaneous administration, sublingual administration, extra-amniotic administration, intra-arterial administration, intra-articular administration, intracardiac administration, intracavernous administration, intralesional administration, subcutaneous administration, intradermal administration, intralesional administration, intramuscular administration, intraosseous administration, intraperitoneal administration, intrathecal administration, intrauterine administration, intravaginal administration, intravesical administration, intravitreal administration, subcutaneous administration, transdermal administration, perivascular administration, and transmucosal administration. In particular, we include methods of administration including but not limited to epidural injection, intracerebral injection, intracerebroventricular injection, sublingual injection, extra-amniotic injection, intra-arterial injection, intra-articular injection, intracardial injection, intrapericardial injection, intracavernous injection, subcutaneous injection, intradermal injection, intramuscular injection, intraosseous injection, intraperitoneal injection, intrathecal injection, intrauterine injection, intravesical injection, intravitreal injection, subcutaneous injection, and perivascular injection.
The mucin-binding targeting agents of the invention and/or compositions comprising such agents may be for use as a medicament.
The mucin-binding targeting agents of the invention and/or compositions comprising such agents may be for use in the treatment of a disease, illness, or disorder in a subject.
Disease, illness, or disorder to be treated may be selected from the group of inflammatory, immunological, endocrine, or metabolic disorders such as obesity or may be neurological, psychological or psychiatric or mood disorders, or disorders of the nervous system, or sexual disorders including reproductive disorders and disorders of the genital system , or may be neoplastic disorders such as cancers. Also contemplated are disorders involving dysfunction of mucous tissue or dysfunction of epithelial tissue, including disorders, diseases, and illnesses of the gastrointestinal tract, nasal disorders, disorders and diseases of the eye, myopathy, obesity, anorexia, weight maintenance, diabetes, disorders associated with mitochondrial dysfunction, genetic disorders, cancer, heart disease, inflammation, disorders associated with the immune system, infertility, disease associated with the brain and/or metabolic energy levels.
Provided herein are also methods of delivery of one or more payloads to a tissue in a subject, wherein the tissue expresses one or more of MUC2, MUC5AC, MUC5B, MUC21. Such methods comprise administering to the subject a pharmaceutical composition comprising a mucin-binding targeting agent in the form of a peptide as provided herein and a payload bound to the polypeptide. The tissue may be located in the gastrointestinal tract, or may be epithelial or non-epithelial tissue located elsewhere. It is contemplated that the binding of the payload to the agent may be via the binding moiety. The binding moiety may be able to release the payload in vivo, such as at the target tissue, e.g. by being able to undergo acid hydrolysis or cleavage by an enzyme such as a protease.
The mucin-binding targeting agents described herein are advantageous in that they display a very distinct selectivity for specific mucins which allows precision targeting to desired tissues with reduced off-targeting and therefore less adverse effects. In particular, the mucin-binding targeting agents have low affinity for MUC1 compared to desired mucins, such as MUC5AC which is highly expressed in the gastrointestinal tract and the respiratory mucosal surfaces.
Additionally, the mucin-binding targeting agents display very high affinity for selected mucins that are orders of magnitude higher than traditional glycan-binding proteins, including lectins, that bind to sugars. This strong interaction with the target mucin facilitates prolonged retention of the mucin-binding targeting agent and any associated payload at a desired site of action.
The mucin-binding targeting agents of the invention may comprise an isolated X409 peptide according to SEQ ID NO:1, or a sequence having a certain sequence identity thereto. Such sequence identity may be e.g. 65% or more, such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more. Sequence identity may be calculated using techniques known in the art, such as those of Example 4.
In some embodiments of the invention, the mucin-binding targeting agent consist of the isolated X409 peptide according to SEQ ID NO:1 or a sequence with at least 75% sequence identity to SEQ ID NO:1, such as at least 80% sequence identity to SEQ ID NO:1, such as at least 90% sequence identity to SEQ ID NO:1, such as at least 95% sequence identity to SEQ ID NO:1.
The X409 peptide may come from different bacterial source as this particular domain shares a high degree of homology across species. Thus, an embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide comprises an X409 peptide according to SEQ ID NO:1 or a X409 peptide derived from E. coli, A. Hydrophilia, or S. baltica having at least 75% sequence identity to SEQ ID NO: 1.
Another embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide is a X409 peptide selected from the group consisting of:
i) SEQ ID NO: 1, SEQ ID NO: 73, SEQ ID NO:74, and SEQ ID NO:135, and
ii) isolated peptides comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 73, SEQ ID NO:74, or SEQ ID NO:135.
A preferred embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide comprises SEQ ID NO:135 (E. coli (accession number AUM10835)) or an isolated peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:135.
Another preferred embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide comprises SEQ ID NO:73 (Shewanella baltica OS223 (accession number ACK48812)) or an isolated peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:73.
A further preferred embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide comprises SEQ ID NO:74 (Aeromonas hydrophilia (accession number QBX76946)) or an isolated peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74.
The mucin-binding targeting agents of the invention may comprise an isolated X409 peptide according to any one of SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:135, or a sequence having a certain sequence identity thereto. Such sequence identity may be e.g. 65% or more, such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more.
An embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide is a X409 peptide selected from the group consisting of:
i) SEQ ID NO: 100, SEQ ID NO: 109, SEQ ID NO: 165, SEQ ID NO: 174, SEQ ID NO:450, SEQ ID NO:609, SEQ ID NO:906, SEQ ID NO: 1066, and
ii) isolated peptides comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 100, SEQ ID NO: 109, SEQ ID NO: 165, SEQ ID NO: 174, SEQ ID NO:450, SEQ ID NO:609, SEQ ID NO:906, and SEQ ID NO: 1066.
Another embodiment of the present invention relates to the mucin-binding targeting agent, wherein the isolated peptide comprises SEQ ID NO:109 (Vibrio anaquillarum (Gene Bank accession number AZS25716)) or an isolated peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:109.
It is to be understood, that these mucin-binding targeting agents may comprise or be attached to a payload as described herein. This can be in form of a fusion protein or as a conjugate or complex with another particle or vehicle, such as a lipid particle.
The mucin-binding targeting agents of the invention may alternatively comprise an isolated peptide according to any one of SEQ ID NO: 2 to 5, or a sequence having a certain sequence identity thereto. Such sequence identity may be e.g. 65% or more, such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more. Sequence identity may be calculated using techniques known in the art, such as those of Example 4.
HEK293WT (ECACC 85120602) and all isogenic clones were cultured in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and 2 mM GlutaMAX (Gibco) in a humidified incubator at 37° C. and 5% CO2. The glycoengineered isogenic HEK293 cells used in this study part of the previously reported cell-based glycan array resource36,37.
CRISPR/Cas9 KO was performed using the GlycoCRISPR resource containing validated gRNAs libraries for targeting of all human glycosyltransferases47, and site-directed KI was performed using a modified ZFN ObLigaRe targeted KI strategy, as previously described48,49. In brief, HEK293 cells grown in 6-well plates (NUNC) to ˜70% confluency were transfected for CRISPR/Cas9 KO with 1 mg of gRNA and 1 mg of GFP-tagged Cas9-PBKS and for targeted KI with 0.5 mg of each ZFN-tagged with GFP/Crimson targeted to the safe-harbor AAVS1 site and 1 ug of respective donor plasmid. 24 h post-transfection, cells were bulk-sorted based on GFP expression by FACS (SONY SH800). After one week of culture, the bulk-sorted cells were single cell-sorted into 96-well plates and KO clones were screened for by Indel Detection by Amplicon Analysis (IDAA) as described50, and gene KO of final clones was verified by Sanger sequencing. The allelic insertion status of KI clones was screened by junction PCR with a primer pair covering the junction area between donor plasmid and the AAVS1 locus, and a primer pair flanking the targeted KI locus.
Transmembrane and secreted mucin TR reporter expression constructs were designed as previously described by use of exchangeable inserts of 150-200 amino acids derived from the TR regions of human mucins (
Transient Transfection with Mucin TR Reporters
Transmembrane GFP-tagged mucin TR reporter constructs were transiently expressed in engineered HEK293 cells. Briefly, cells were seeded in 24-wells (NUNC) and transfected at ˜70% confluency with 0.5 μg of plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer's protocol. Cells were harvested 24 h post-transfection and used for assays followed by flow cytometry analysis.
The secreted reporter constructs were stably expressed in isogenic HEK293-6E cell lines selected by two weeks of culture in the presence of 0.32 μg/mL G418 (Sigma-Aldrich) and two rounds of FACS enrichment for GFP expression. A stable pool of cells was seeded at a density of 0.25×106 cells/ml and cultured for 5 days on an orbital shaker in F17 medium (Gibco) supplemented with 0.1 Kolliphor P188 (Sigma-Aldrich) and 2% Glutamax. Culture medium containing secreted mucin TR reporter was harvested (3,000×g, 10 min), mixed 3:1 (v/v) with 4× binding buffer (100 mM sodium phosphate, pH 7.4, 2 M NaCl), and run through a nickel-nitrilotriacetic acid (Ni-NTA) affinity resin column (Qiagen), pre-equilibrated with washing buffer (25 mM sodium phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole). The column was washed multiple times with washing buffer and mucin TR reporter was eluted with 200 mM imidazole. Eluted fractions were analyzed by SDS-PAGE and fractions containing the mucin TR reporter were desalted followed by buffer exchange to MiliQ using Zeba spin columns (Thermo Fisher Scientific). Yields were quantified using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer's instructions and NuPAGE Novex Bis-Tris (4-12%, Thermo Fisher Scientific) Coomassie blue analysis.
HPLC (C4) purified mucin TR reporters (10 μg) were incubated in 0.1M NaOH and 1M NaBH4 at 45° C. for 16 h. Released O-glycan alditols were desalted by cation-exchange chromatography (Dowex AG 50W 8X). Borate salts were converted into methyl borate esters by adding 1% acetic acid in methanol and evaporated under N2 gas. Desalted O-glycan alditols were permethylated (in 150 μL DMSO, ˜20 mg NaOH powder, 30 μl methyl iodide) at room temperature for 1 h. The reaction was terminated by addition of 200 μL ice-cold MQ water followed by the addition of ˜200 μL chloroform. The organic phase was washed 5 times with 1 mL MQ water and evaporated under N2 gas. Permethylated O-glycans were purified by custom Stage Tips (C18 sorbent from Empore 3 M) and eluted in 20 mL 35% (v/v) acetonitrile, of which 1 μl was co-crystalized with 1 μL DHB matrix (10 mg/ml in 70% acetonitrile, 0.1% TFA, 0.5 mM sodium acetate) before positive mode MALDI-TOF analysis.
Ni-chromatography purified intact mucin TR reporters (50 μg) were digested with 1 μg Lys-C (Roche) at a 1:35 ratio at 37° C. for 18 h in 50 mM ammonium bicarbonate buffer (pH 8.0). After heat inactivation at 98° C. for 15 min, reactions were dried by speed vac and desialylated with 40 mU C. perfringens neuraminidase (Sigma-Aldrich) for 5 hrs at 37° C. in 65 mM sodium acetate buffer (pH 5.0). This step was omitted for reporters expressed in HEK293KO COSMC (Tn glycoforms). Samples were heat inactivated at 98° C. for 15 min and dried. For intact MS analysis samples were separated on by C4 HPLC (Aeris™ C4, 3.6 μm, 200 a, 250×2.1 mm, Phenomenex) using a 0-100% gradient of 90% acetonitrile in 0.1% TFA. Fractions containing the released TR O-glycodomains were verified by ELISA with lectins or mAbs, dried and resuspended in 20 μl of 0.1% FA for intact mass analysis. For bottom up analysis of the MUC1 reporter, samples (20 μg) were further digested 2× with 0.67 μg Endo-AspN at a 1:35 ratio for 18 hrs at 37° C. in 100 mM Tris-HCL (pH 8.0). After inactivation by the addition of 1 mL of concentrated TFA, samples were desalted using custom Stage Tips (C18 sorbent from Empore 3 M) and analyzed by LC-MS/MS.
LC MS/MS analysis was performed on EASY-nLC 1200 UHPLC (Thermo Fisher Scientific) interfaced via nanoSpray Flex ion source to an Orbitrap Fusion Lumos MS (Thermo Fisher Scientific). Briefly, the nLC was operated in a single analytical column set up using PicoFrit Emitters (New Objectives, 75 mm inner diameter) packed in-house with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 1.9-mm particle size, 19-21 cm column length). Each sample was injected onto the column and eluted in gradients from 3 to 32% B for glycopeptides, and 10 to 40% for released and labeled glycans in 45 min at 200 nL/min (Solvent A, 100% H2O; Solvent B, 80% acetonitrile; both containing 0.1% (v/v) formic acid). A precursor MS1 scan (m/z 350-2,000) of intact peptides was acquired in the Orbitrap at the nominal resolution setting of 120,000, followed by Orbitrap HCD-MS2 and ETD-MS2 at the nominal resolution setting of 60,000 of the five most abundant multiply charged precursors in the MS1 spectrum; a minimum MS1 signal threshold of 50,000 was used for triggering data-dependent fragmentation events. Targeted MS/MS analysis was performed by setting up a targeted MSn (tMSn) Scan Properties pane.
Samples were analyzed by EASY-nLC 1200 UHPLC (Thermo Scientific Scientific) interfaced via nanoSpray Flex ion source to an on OrbiTrap Fusion/Lumos instrument (Thermo Scientific Scientific) using “high” mass range setting in m/z range 700-4000. The instrument was operated in “Low Pressure” Mode to provide optimal detection of intact protein masses. MS parameters settings: spray voltage 2.2 kV, source fragmentation energy 35 V. All ions were detected in OrbiTrap at the resolution of 7500 (at m/z 200). The number of microscans was set to 20. The nLC was operated in a single analytical column set up using PicoFrit Emitters (New Objectives, 75 mm inner diameter) packed in-house with C4 phase (Dr. Maisch, 3.0-mm particle size, 16-20 cm column length). Each sample was injected onto the column and eluted in gradients from 5 to 30% B in 25 min, from 30 to 100% B in 20 min and 100% B for 15min at 300 nL/min (Solvent A, 100% H2O; Solvent B, 80% acetonitrile; both containing 0.1% (v/v) formic acid).
Glycopeptide compositional analysis was performed from m/z features extracted from LC-MS data using in-house written SysBioWare software51. For m/z feature recognition from full MS scans Minora Feature Detector Node of the Proteome discoverer 2.2 (Thermo Fisher Scientific) was used. The list of precursor ions (m/z, charge, peak area) was imported as ASCII data into SysBioWare and compositional assignment within 3 ppm mass tolerance was performed. The main building blocks used for the compositional analysis were: NeuAc, Hex, HexNAc, dHex and the theoretical mass increment of the most prominent peptide corresponding to each potential glycosites. Upon generation of the potential glycopeptide list each glycosite was rank for the top 10 most abundant candidates and each candidate structure was confirmed by doing targeted MS/MS analysis followed by manual interpretation of the corresponding MS/MS spectrum. For intact mass analysis raw spectra were deconvoluted to zero-charge by BioPharma Finder Software (Thermo Fisher Scientific, San Jose) using default settings. Glycoproteoforms were annotated by in-house written SysBioWare software51 using average masses of Hexose, N-acetylhexosamine, and the known backbone mass of mucin TR reporter increment (MUC1, MUC2, MUC7, etc)
For lectin staining HEK293 cells transiently expressing mucin TR reporters were incubated on ice or at 4° C. with biotinylated PNA, VVA (Vector Laboratories) or Pan-lectenz (Lectenz Bio) diluted in PBA (1× PBA containing 1% BSA (w/v)) for 1 h, followed by washing and staining with Alexa Fluor 647-conjugated streptavidin (Invitrogen) for 20 min. Stainings with mAbs specific to mucin glycoforms produced in mice was performed by incubating cells for 30 min at 4° C. with supernatant harvested from the respective hybridoma followed by staining with FITC-conjugated polyclonal rabbit anti-mouse Ig (Dako). Cells were stained with GST-tagged streptococcal adhesins at different concentrations diluted in PBA for 1 h on ice, followed by incubation with rabbit polyclonal anti-GST antibodies (Thermo Fisher) for 1 h and subsequent staining with Alexa Fluor 647 conjugated goat anti-rabbit IgG (Thermo Fisher) for 1 h. All cells were resuspended in PBA for flow cytometry analysis (SONY SA3800).
ELISA assays were performed using MaxiSorp 96-well plates (Nunc) coated with dilutions of purified mucin TR reporters starting from 100 ng/ml or fractions derived from C4 HPLC incubated o/n at 4° C. in 50 ml carbonate-bicarbonate buffer (pH 9.6). Plates were blocked with PLI-P buffer (PO4, Na/K, 1% Triton-X100, 1% BSA, pH 7.4) and incubated with mAbs (undiluted culture sups or as indicated) or biotinylated-lectins (Vector Laboratories and Lectenz Bio) for 1 h at RT, followed by extensive washing with PBS containing 0.05% Tween-20, and incubation with 50 ml of 1 ug/mL HRP conjugated anti mouse Ig (Dako) or 1 ug/mL streptavidin-conjugated HRP (Dako) for 1 h. Plates were developed with TMB substrate (Dako) and reactions were stopped by addition of 0.5 M H2SO4 followed by measurement of absorbance at 450 nm.
Recombinant StcE, StcEE447D, StcEΔX409, and X409 were produced in E. coli similarly as reported previously24. Enzyme assays with purified intact mucin reporters (500 ng) were performed by incubating serial dilutions of StcE for 2 h at 37° C. in 20 mL reactions in 50 mM ammonium bicarbonate buffer, and reactions were stopped by heat-inactivation at 95° C. for 5 min. Samples were run on NuPAGE Novex gels (Bis-Tris 4-12%) at 100 V for 1 h followed by staining with Krypton Fluorescent Protein Stain (Thermo Fisher Scientific) according to the manufacturer's instructions. Gels were imaged using an ImageQuant LAS 4000 system (GE Healthcare). Cell-based activity assays were performed with HEK293 cells transiently expressing mucin TR reporters incubated with serial dilutions of StcE in PBA at 37° C. After 1 h, cells were washed with PBA, stained with APC-conjugated anti-FLAG antibody (BioLegend) for 30 min at 4° C., and washed cells were analyzed by flow cytometry. Mean fluorescent intensity of anti-FLAG binding to GFP positive (transfected) and negative (untransfected) populations was quantified as using FlowJo (FlowJo LLC). For cell-binding assays with X409-GFP, HEK293 cells expressing CFP-tagged membrane TR reporters were used. Cells were incubated with different concentrations of X409-GFP for 1 h at 4° C. followed by staining with APC-conjugated anti-FLAG antibody. X409-GFP binding to anti-FLAG positive cells was quantified using FlowJo software. For histology analysis, deparrafinized tissue microarray sections52 were microwave treated for 20 min in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval followed by 1 h blocking with 1× PBS containing 5% BSA (w/v). Sections were incubated o/n at 4° C. with 5 μg/ml 6xHis-tagged StcE or StcEΔX409 followed by washing and subsequent 1 h incubation with first mouse anti-6xHis antibody (Thermo Fisher) and second AF488-conjugated rabbit anti-mouse IgG (Invitrogen).
Sections stained with 2 μg/ml GFP-X409 were optionally sialidase treated and co-stained with mouse anti-MUC2 (PMH1) and donkey-anti mouse IgG Cy3 (Jackson ImmunoResearch). All samples were mounted with ProLong Gold Antifade Mountant with DAPI (Molecular Probes) and imaged using a Zeiss microscopy system followed by analysis with ImageJ (NIH).
Data analysis
Glycopeptide compositional analysis was performed from m/z features extracted from LC-MS data using in-house written SysBioWare software51. For m/z feature recognition from full MS scans Minora Feature Detector Node of the Proteome discoverer 2.2 (Thermo Fisher Scientific) was used. The list of precursor ions (m/z, charge, peak area) was imported as ASCII data into SysBioWare and compositional assignment within 3 ppm mass tolerance was performed. The main building blocks used for the compositional analysis were: NeuAc, Hex, HexNAc, dHex and the theoretical mass increment of the most prominent peptide corresponding to each potential glycosite. Upon generation of the potential glycopeptide list each glycosite was ranked for the top 10 most abundant candidates and each candidate structure was confirmed by doing targeted MS/MS analysis followed by manual interpretation of the corresponding MS/MS spectrum. For intact mass analysis raw spectra were deconvoluted to zero-charge by BioPharma Finder Software (Thermo Fisher Scientific, San Jose) using default settings. Glycoproteoforms were annotated by in-house written SysBioWare software51 using the average masses of Hexose, N-acetylhexosamine, and the known backbone mass of mucin TR reporter increment.
The purpose of the following examples is given as an illustration of various aspects of the invention and are thus not meant to limit the present invention in any way. Along with the present examples the methods described herein are presently representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1—Engineering Strategy for Display of the Human Mucinome
For long mucins have represented a black box in exploring the molecular cues that serve in intrinsic interactions with glycan-binding proteins and in extrinsic interactions with microorganisms53. Dissection of interactions with simple O-glycan structures found on mucins have benefited tremendously from the development of printed glycan arrays43,54, and these have for decades served as essential tools in exploring the interactome of glycans and proteins41. However, mucins and their large variable TR domains present O-glycans in different densities, and patterns are likely to provide more specific interactions and instructive cues. Mucin TRs differ markedly in sequence, length and numbers within closely related mammals9, and this divergence in TRs may have evolved to accommodate specific recognition of higher order patterns and clusters of O-glycans36. We previously provided evidence for this by use of the cell-based glycan array demonstrating that two distinct streptococcal Siglec-like adhesins bind selectively to O-glycans presented on distinct mucin-like domains in O-glycoproteins and mucins36.
Cell-based display strategies allow for presentation of glycans in the natural context of glycoproteins and the cell surface, and this has provided the first experimental evidence for the existence of higher order binding motifs consisting of O-glycans in dense patterns36. We hypothesized that the main cues for the microbiota lie in the TR regions that display O-glycans of diverse structures and position in unique patterns. The traditional interpretation is that the divergence in TR sequences has co-evolved with the microbiota to govern refined interactions with larger motifs of O-glycan patterns as recently suggested for streptococcal serine-rich adhesins36. The TR regions of mucins are quite distinct in length and in sequences with distinct spacing of O-glycosites55, and TRs in any mucin exhibit individual variability in numbers as well as to some degree in actual sequences10. Thus, there are rich opportunities for unique codes in mucin TRs, governed by the particular display of patterns and structures of O-glycans. The mucin TRs and their glycocodes may be considered as the informational content of mucins and thus comprise the mucinome. The TR mucinome provides a much greater potential binding epitome than the comparatively limited repertoire of binding epitopes comprised of simple oligosaccharide motifs available in humans42.
We therefore sought to capture the molecular cues contained in human mucin TRs and enable molecular dissection of these cues. We developed a cell-based platform for display and production of representative mucin TRs with defined O-glycans. We reasoned that most of the features of human mucin TRs could be displayed in shorter segments of 150-200 amino acids, and used a GFP-tagged expression construct design containing representative TRs from different mucins to produce a library of cell membrane and secreted mucin TR reporters in human embryonic kidney (HEK293) cells with distinct programmed O-glycosylation capacities. Strikingly, we found that these mucin TR reporters could readily be produced as highly homogeneous molecules with essentially complete O-glycan occupancies and with distinct O-glycan structures in amounts that enabled us to characterize the simplest reporters by intact mass spectrometry (MS), and hence circumvent the longstanding obstacles with protease digestion and bottom-up analysis of mucins14,15. We demonstrate that the cell-based mucin display can be used to produce and display defined mucin TRs and mucin-like O-glycodomains with custom designed O-glycosylation.
In
The transmembrane TR reporters were expressed transiently in glycoengineered HEK293 cells that do not appear to express endogenous mucins, and the secreted reporters were expressed stably36,56 We took advantage of our previously reported O-glycoengineering strategy to establish designs for homogeneous O-glycosylation capacities that result in attachment of defined O-glycan structures (
To validate the cell-based mucin TR display platform we first used immunocytology analyses. We previously verified the general glycosylation outcomes of most of the glycoengineering performed in HEK293 cells36. We therefore tested a subset of transiently expressed membrane bound TR reporters with lectins and monoclonal antibodies (mAbs) with well characterized specificities for distinct O-glycan structures (
We also probed the mucin TR reporters with a panel of mAbs directed to human mucin TR regions, most of which are known to be affected by O-glycosylation either because glycosylation interferes with or blocks binding to the protein core (e.g. mAb to MUC1 such as SM3 or 5E10)58 or because O-glycans are required for the binding (e.g. mAbs to Tn-MUC1 (5E5), Tn-MUC2 (PMH1), and Tn-MUC4 (3B11)59-61 (
We further performed structural analysis of the isolated secreted mucin TRs. Secreted TR reporters stably expressed in glycoengineered HEK293 cells were isolated by Ni-chromatography and assessed by SDS-PAGE analysis, which showed that the GFP-tagged proteins migrated as distinct rather homogeneous bands (
The dense O-glycosylation of mucin TRs in most cases blocks cleavage by peptidases limiting conventional glycoproteomics strategies14,16. However, the MUC1 TRs are cleavable by endoproteinase-Asp-N (AspN) in the PDTR sequence17, 18,19 and we therefore used the MUC1 reporter for full characterization (
The promising results obtained with intact MS analysis of the MUC1 TR glycodomains prompted intact MS analysis of the simplest Tn glycoforms of MUC2, MUC5AC, MUC7, MUC13, and MUC21 TR glycodomains, which showed similarly high occupancy of available glycosites with rather homogeneous patterns (
Example 2—Applying the Cell-based Mucin Array for Analysis of the Cleavage Activity of the Glycoprotease StcE
Cell-based display strategies allow for presentation of glycans in the natural context of glycoproteins and the cell surface, and this has provided the first experimental evidence for the existence of higher order binding motifs consisting of O-glycans in dense patterns 36. We hypothesized that the main cues for the microbiota lie in the TR regions that display O-glycans of diverse structures and position in unique patterns. The traditional interpretation is that the divergence in TR sequences has co-evolved with the microbiota to govern refined interactions with larger motifs of O-glycan patterns as recently suggested for streptococcal serine-rich adhesins36. The TR regions of mucins are quite distinct in length and in sequences with distinct spacing of O-glycosites55, and TRs in any mucin exhibit individual variability in numbers as well as to some degree in actual sequences10. Thus, there are rich opportunities for unique codes in mucin TRs, governed by the particular display of patterns and structures of O-glycans. The mucin TRs and their glycocodes may be considered the informational content of mucins and thus comprise the mucinome. The TR mucinome provides a much greater potential binding epitome than the comparatively limited repertoire of binding epitopes comprised of simple oligosaccharide motifs available in humans42.
We therefore sought to capture the molecular cues contained in human mucin TRs and enable molecular dissection of these cues. We developed a cell-based platform for display and production of representative mucin TRs with defined O-glycans. We reasoned that most of the features of human mucin TRs could be displayed in shorter segments of 150-200 amino acids, and used a GFP-tagged expression construct design containing representative TRs from different mucins to produce a library of cell membrane and secreted mucin TR reporters in human embryonic kidney (HEK293) cells with distinct programmed O-glycosylation capacities. Strikingly, we found that these mucin TR reporters could readily be produced as highly homogeneous molecules with essentially complete O-glycan occupancies and with distinct O-glycan structures in amounts that enabled us to characterize the simplest reporters by intact mass spectrometry (MS), and hence circumvent the longstanding obstacles with protease digestion and bottom-up analysis of mucins14,20. We demonstrate that the cell-based mucin display can be used to produce and display defined mucin TRs and mucin-like O-glycodomains with custom designed O-glycosylation.
Availability of the cell-based mucin display platform enabled for the first time detailed analysis of the substrate specificities of microbial glycopeptidases such as the StcE glycoprotease. Moreover, using this platform we were able to demonstrate that StcE cleaved several different types of mucins, but not MUC1 as previously indicated23. Moreover, we could demonstrate that StcE efficiently cleaves mucin TRs with different types of O-glycans including core1 and core2 O-glycans, but importantly StcE cleavage is blocked by core3 and sialyl-Tn O-glycans found predominantly in the human intestine.
The mucin display platform is ideal for discovery and exploration of mucin degrading enzymes such as the pathogenic glycoprotease StcE21-24. EHEC is a food-derived human pathogen able to colonize the colon and cause gastroenteritis and bloody diarrhea. Strains of the O157:H7 serotype carry a large virulence plasmid pO157:H7 that directs secretion of StcE21,26. StcE is predicted to provide EHEC with adherence to the gastrointestinal tract and ability to penetrate through the mucin layers via its impressive mucin degrading properties64. StcE cleaves the C1 esterase inhibitor glycoprotein (C1-INH) that contains a highly O-glycosylated mucin-like domain and is required for complement activation21. StcE was previously shown to cleave several mucins including MUC1, MUC7 and MUC1622,23,25,65, and the cleavage required O-glycosylation and accommodated complex O-glycan structures23. The gut microbiome is contained in a network of the gel forming mucin MUC2 that forms the loose outer mucin layer, and a dense inner layer of MUC2 forms a barrier and prevents the microbiota to reach the underlying colonic epithelium66,67. Cleavage of the MUC2 mucin layers by StcE would destroy the important barrier function.
We used purified TR reporters and those displayed on cells to further explore the fine substrate specificity of recombinant purified StcE glycoprotease and to dissect its reported mucin-binding properties (
The cell-based mucin display platform presented here offers a unique resource with wide applications and opportunities for discovery and dissection of molecular properties of natural human mucins and other glycoproteins with mucin-domains. The informational cues harbored in mucin TRs with their distinct patterns and structures of O-glycans can be addressed with well-defined molecules in a variety of assay formats. This was illustrated by our use of the mucin display to dissect the fine substrate specificity of the mucin-destroying glycoprotease StcE derived from pathogenic EHEC21,22, demonstrating clear selectivity for both distinct mucin TRs and O-glycoforms, and importantly discovering that the normal core3 O-glycosylation pathway in colon actually inhibits StcE digestion of MUC2.
Example 3—Analysis of the Tissue Binding Properties of the Glycoprotease StcE
By close examination of the 3-D structure of StcE24 we noticed that the protein contained a C-terminal domain opposite to the catalytic metalloprotease domain (M66), and we hypothesized that this could have a function for StcE. The small domain is a peptide of approximately 100 amino acids, and by detailed sequence analysis we predicted that this could represent an evolutionarily mobile binding module (here designated X409). To test the potential function of the X409 module we first analyzed if deleting this domain affected the mucin cleaving function of StcE using the cell-based mucin display platform. Surprisingly, we found that StcE without X409 retained its remarkable ability to cleave mucin TRs. We therefore next considered whether the module had a role in the mucin binding properties of StcE, which was previously demonstrated with the catalytically inactivated StcEE447D mutant27. We first tested the wildtype StcE active enzyme in binding to human mucosal tissues, and surprisingly found that wildtype StcE without the inactivating mutation bound mucin producing cells similar to the StcEE447D mutant (
Detailed analysis of the binding pattern with the mucin TR display suggested that a common feature for X409 binding was a sequence motif of 5-6 clustered O-glycans. To test this hypothesis not only by the cell-based human mucin TRs, we tested isolated commercial animal mucins (BSM, PSM, and OSM) by ELISA. Only PSM is known to contain such long clusters of O-glycans, and X409 exhibited binding only to PSM and not to BSM and asialo OSM (
The small X409 peptide module offers a unique molecule for binding to select mucins. We show that a fusion protein comprising X409 can be used to bind gastric and intestinal mucosa, and this offers an elegant way to deliver and retain molecules at such anatomical sites for diagnostic and therapeutic purposes.
StcE plays a role in adherence of EHEC to the intestinal epithelium by binding mucins22,24,28, and a catalytically inactive mutant of StcE (StcEE447D) exhibits broad binding to mucin producing cells in tissue sections27,24. Examination of the 3-D structure of StcE revealed that the protein contained a C-terminal domain (here designated X409) opposite to the catalytic metalloprotease domain (M66). The X409 module was predicted to be important for the catalytic function of StcE, and we therefore produced a StcE mutant construct without this domain (StcEΔX409) (
We further tested the affect O-glycan structures on the mucin TRs have on the binding of X409, and surprisingly the strong binding to MUC2 and MUC5AC was only slightly influenced by the O-glycan structures attached to the TRs, although weaker binding to TRs carrying Tn, core3 and especially STn O-glycans was observed (
The unique binding properties of X409 to select mucins independent of the structure of the attached O-glycans (
The binding affinity of traditional lectins to glycans is typically weak (Kd=10−3-10−6 M, mM to μM) in comparison to binding affinity of antibody-antigen interactions that can be <10−9 M (<nM), although increased binding affinity of lectins to glycans may be observed with multivalent interactions. The unique mucin-binding properties of X409 with select binding to distinct mucins and without select binding to distinct O-glycan structures prompted testing of the binding affinity and mode of binding to a mucin reporter with different defined O-glycans attached (
The high affinity binding properties of X409 to select mucins with elaborated mature O-glycans is unique and dissimilar to traditional lectins and glycan-binding proteins with low affinity. Moreover, the select binding to distinct mucins and not for example MUC1 strongly indicate that X409 has unique binding properties and recognize a motif comprised of the innermost part of multiple O-glycans attached to the mucin protein backbone. A similar high affinity binding to glycosylated mucins have only been described for monoclonal antibodies to the cancer-associated Tn glycoform of MUC1 (Tn-MUC1), which recognize the glycans and part of the protein backbone. Thus, the X409 mucin-binding module represent a new class of non-immunoglobulin binders that have selectivity for distinct glycoproteins and high affinity binding like antibodies while relying on glycans for binding similar to lectins.
Example 4—The family of X409 mucin-binding modules. The 3-D structure of StcE24 was used to identify the boundaries of the X409 mucin-binding module from Escherichia coli O157:H7 (
To further validate that the identified X409 related sequences represents genuine functional mucin-binding modules, four predicted X409 modules were selected from the wide range of identified X409 related sequences (
Shown in
Notably, binding to the most immature glycoforms is preferable for example when using the mucin-binding properties of X409 to target or deliver substances to the most nascent mucin layers in mucosal linings. Binding to the immature glycoforms of mucins is for example not preferable when using the mucin-binding properties of X409 to target or deliver substances to mucosal linings such as the gut where bacteria continuously degrade the glycans resulting in appearance of for example Tn glycoforms at the most superficial mucus layers and in shed mucins. Thus, the ability of X409 variants to selectively (StcE X409 and other variants) or exclusive (E. coli X409 accession number AUM10835) is preferable to target deeper mucus layers in the gut mucosa and prevent adherence to the superficial and shed mucin layers. All four modules exhibit selective mucin-binding preference for the mucin MUC5Ac (HEK293 cells displaying the MUC5Ac reporter with core1 T O-glycans) similarly to StcE X409 (
Example 5—Novel families of mucin-binding modules unrelated to the X409 sequence. We next searched for CBM-related modules associated with peptidases. The CBM families that were searched were those listed in the Carbohydrate-Active Enzymes database www.cazy.org;75 BlastP73 was used to identify proteins with similarity to representative CBM sequences in the non-redundant protein sequence database of the NCBI using default parameters, followed by the identification of domains related to peptidases using Pfam74 using default parameters. This resulted in identification of diverse sequence modules with sequences unrelated to X409, but with similar characteristics of distinct modules positioned N- or C-terminal to predicted peptidase domains (
A detailed analysis of the mucin-binding properties of the novel modules were performed by flow cytometry with HEK293 cells displaying mucin reporters with three different O-glycan structures (Disialyl-core2, core1 T, and Tn) (
These results clearly demonstrate that unique mucin-binding modules can be identified among X409 unrelated sequences, and that these have different mucin-binding properties with selectivity for different mucins and O-glycan structures. Thus, the X409-related and unrelated mucin-binding modules presented here offers a variety of targeting modules characterized by their unique binding properties for select mucins and O-glycans that are useful for targeting and delivery.
Example 6—Use of Mucin-binding Modules to Target/Deliver to Mucosal Surfaces
The mucin-binding modules are valuable for targeting and delivery of substances to mucosal surfaces and the distinct mucin and glycan binding properties of the X409-related and unrelated mucin-binding modules can be used to custom-design and tune targeting to different mucosal surfaces expressing different mucins and O-glycans. The mucin-binding modules can for example be used to target orally delivered substances to the gut mucosa, inhaled substances to the respiratory mucosa, and topically administered substances to for example the vaginal and nasal lining mucosa. Preferable substances for delivery to mucosal surfaces include bioactive peptides including peptide hormones, small molecule drugs, enzymes, vaccine compositions, RNA and DNA. Delivery of enzymes may include therapeutic enzymes, for example digestive enzymes needed following surgical removal of pancreas, and enzymes used for improving feed digestion and uptake of nutrients.
The mucin-binding modules are used in construction of conjugates, complexes and lipid particles where the mucin-binding module is chemically linked, complexed with, adsorbed to, or incorporated into for example lipid particles. For example by chemical conjugation of a lipid moiety to the mucin-binding module this lipid modified module is inserted into lipid particles that may contain substances, and such coated particles adhere to and are retained at the mucosal surface.
The mucin-binding modules are used in chimeric fusion protein designs where the mucin-binding module is added to a bioactive protein, such as therapeutic protein, bioactive peptide, or enzyme, by recombinant gene technologies to for example mediate binding to the mucosa and enhance residence time, biodistribution, and bioactive effects. The mucin-binding modules are for example used to enhance efficiency of enzymes like proteases, lipases, phytases, amylase, xylanases, β-Glucanases, α-Galactosidases, mannanases, cellulases, hemicellulases, and pectinases.
One example of delivery of fusion protein drug is protein sequences built on a rigid alpha-helicoidal HEAT-like protein sequences (αReps) that recognize the SARS-COV-2 spike receptor ACE2 binding domain and neutralize virus infection. Installation of such αReps in the nasal cavity before or during infections effectively reduce the replication of a SARS-COV-2 strain in the nasal epithelium in hamsters. The αRep protein localize to the surface mucosa and is detectable for 0-30 min by immunohistological analysis using antibodies to tags, but at 60 min the protein is barely detectable. By introducing X409 to the αRep protein the chimeric fusion protein will remain detectable at the surface considerably longer (for example up to 6 hours) after nasal instillation and thereby provide longer bioactivity and inhibition of viral infection. For example the αRep F9-C2 protein or X409 fusion protein hereof may be given as a prophylactic to limit SARS-COV-2 infection in vivo. Syrian golden hamsters that reflect the infection in human may be pretreated with for example 0.6 mg of the proteins distributed between the two nostrils 1h prior to infection with SARS-COV-2, and the presence of infiltrated αReps on the surface epithelium layer will be observed indicating an efficient absorption of the molecule.
The X409 mucin-binding module is further useful for improving mucosal vaccine delivery and effectiveness. Mucosal vaccine formulations are dependent on uptake and presentation by resident mucosal innate immune cells and antigen presenting cells, and topical or inhaled (for example by spray) formulations of vaccines are improved by extending their residence time at the mucosal surface. The X409 mucin-binding module is useful to attach by covalent or non-covalent methods to a vaccine formulation or in chimeric fusion protein designs of vaccines comprising for example recombinant proteins or included in the coding region of RNA and DNA vaccine designs to improve adhesion to oral, nasal and other respiratory mucosal surfaces and significantly enhance residence time and effectiveness. The X409 module may be incorporated in RNA and DNA vaccine designs by introducing the coding region for X409 as outlined in this invention in the design (for example before, after, or separate) to the coding region for the protein immunogen of interest. The X409 module may also be conjugated and/or incorporated in the delivery vehicle for mucosal RNA and DNA vaccine formulations to allow the formulation to adhere and reside for extended periods at the mucosal surface for effective delivery of vaccines. The X409 module may also be used for protein, glycoprotein and polysaccharide vaccines by conjugating, incorporating and/or fusing the X409 module to recombinant vaccines in order to enhance mucosal adhesions and effectiveness.
<110> University of Copenhagen
<120> PEPTIDES WITH MUCIN-BINDING PROPERTIES
<130> P77084EP
<160> 5
<170> BISSAP 1.3.6
<210> 1
<211> 93
<212> PRT
<213>Escherichia coli
<223> X409 module of StcE
<210> 2
<211> 153
<212> PRT
<213> Clostridium perfringens
<223> HC1 CBM51
<210> 3
<211> 276
<212> PRT
<213> Bacillus cereus
<223> HC7 X408-FN3-CBM5 of K8
<210> 4
<211> 336
<212> PRT
<213> Bacteroides fragilis
<223> HC11 Bacon-Bacon-CBM32
<210> 5
<211> 237
<212> PRT
<213> Bacteroides fragilis
<223> HC12 Bacteroides thetaiotaomicron
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X1. A mucin-binding targeting agent comprising an isolated peptide selected from the group comprising
X409 peptide according to SEQ ID NO: 1,
HC1 CBM51 peptide according to SEQ ID NO: 2,
HC7 X408-FN3-CBM5 peptide according to SEQ ID NO:3,
HC11 Bacon-Bacon-CBM32 peptide according to SEQ ID NO: 4, and
HC12 Bacteroides thetaiotaomicron peptide according to SEQ ID NO: 5;
or a mucin-binding targeting agent having 80% sequence identity or more to any one of SEQ ID NO: 1 to 5.
X2. The mucin-binding targeting agent according to item X1 wherein the isolated peptide has a sequence identity of 95% or more to any one of SEQ ID NO:1 to 5.
X3. The mucin-binding targeting agent according to any one of items X1 or X2 wherein the peptide is catalytically inactive against mucins.
X4. The mucin-binding targeting agent according to any one of items X1 to X3 wherein the mucin to which the mucin-targeting agent binds is one or more of MUC2, MUC5AC, MUC5B, and MUC21.
X5. The mucin-binding targeting agent according to any one of items X1 to X4 further comprising a binding moiety.
X6. The mucin-binding targeting agent according to any one of items X1 to X5 further comprising a payload.
X7. The mucin-binding targeting agent according to items X5 or X6 wherein the payload is attached to the agent via the binding moiety.
X8. The mucin-binding targeting agent according to any one of items X5 to X7 wherein the binding moiety is selected from the group comprising esters, lipid anchors, biotin, streptavidin, antibodies, nanobodies, and peptide linkers.
X9. The mucin-binding targeting agent according to any one of items X6 to X8, wherein the payload is selected from the group comprising a therapeutic agent, a detectable marker, nanoparticle, liposome, vesicle and a stain.
X10. The mucin-binding targeting agent according to any of items X1-X9 for use as a medicament.
X11. A composition comprising the mucin-binding targeting agent according to any of items X1-X10.
X12. The composition according to item X11, wherein the composition is a pharmaceutical dosage form further comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable carrier.
X13. The mucin-binding targeting agent according to any of items X1-X10 for use in the treatment of a disease, illness, or disorder in a subject, wherein the disease, illness, or disorder is selected from the group of metabolic, endocrine, inflammatory, immunological diseases, illnesses, or disorders, or is a cancer or a neoplasia.
X14. A method of delivery of a payload to a tissue in a subject, said tissue expressing one or more of MUC2, MUCSAC, MUC5B, and MUC21, said method comprising administering to the subject a pharmaceutical composition comprising a mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1;
HC1 CBM51 peptide according to SEQ ID NO: 2;
HC7 X408-FN3-CBM5 peptide according to SEQ ID NO:3;
HC11 Bacon-Bacon-CBM32 peptide according to SEQ ID NO: 4; and
HC12 Bacteroides thetaiotaomicron peptide according to SEQ ID NO: 5;
or a mucin-binding targeting agent comprising a sequence having 80% sequence identity to any one of SEQ ID NO: 1 to 5, and a payload bound to said peptide.
X15. A method of preparing a mucin-binding targeting agent, the method comprising the step of providing an isolated
X409 peptide according to SEQ ID NO: 1;
HC1 CBM51 peptide according to SEQ ID NO: 2;
HC7 X408-FN3-CBM5 peptide according to SEQ ID NO:3;
HC11 Bacon-Bacon-CBM32 peptide according to SEQ ID NO: 4; and
HC12 Bacteroides thetaiotaomicron peptide according to SEQ ID NO: 5;
or a mucin-binding targeting agent having 80% sequence identity to any one of SEQ ID NO: 1 to 5.
1. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 65% sequence identity or more thereto.
2. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 75% sequence identity or more thereto.
3. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 80% sequence identity or more thereto.
4. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 85% sequence identity or more thereto.
5. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 90% sequence identity or more thereto.
6. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 95% sequence identity or more thereto.
7. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 96% sequence identity or more thereto.
8. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 97% sequence identity or more thereto.
9. A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 98% sequence identity or more thereto.
10 A mucin-binding targeting agent comprising an isolated X409 peptide according to SEQ ID NO: 1 or a sequence having 99% sequence identity or more thereto.
11. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 65% sequence identity or more thereto.
12. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 75% sequence identity or more thereto.
13. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 80% sequence identity or more thereto.
14. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 85% sequence identity or more thereto.
15. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 90% sequence identity or more thereto.
16. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 95% sequence identity or more thereto.
17. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 96% sequence identity or more thereto.
18. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 97% sequence identity or more thereto.
19. A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 98% sequence identity or more thereto.
20 A mucin-binding targeting agent comprising an isolated HC1 CBM51 peptide according to SEQ ID NO: 2 or a sequence having 99% sequence identity or more thereto.
3:
31. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 65% sequence identity or more thereto.
32. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 75% sequence identity or more thereto.
43. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 80% sequence identity or more thereto.
44. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 85% sequence identity or more thereto.
45. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 90% sequence identity or more thereto.
46. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 95% sequence identity or more thereto.
47. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 96% sequence identity or more thereto.
48. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 97% sequence identity or more thereto.
49. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 98% sequence identity or more thereto.
50. A mucin-binding targeting agent comprising an isolated HC7 X408-FN3-CBM5 peptide according to SEQ ID NO: 3 or a sequence having 99% sequence identity or more thereto.
4:
51. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 65% sequence identity or more thereto.
52. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 75% sequence identity or more thereto.
53. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 80% sequence identity or more thereto.
54. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 85% sequence identity or more thereto.
55. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 90% sequence identity or more thereto.
56. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 95% sequence identity or more thereto.
57. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 96% sequence identity or more thereto.
58. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 97% sequence identity or more thereto.
59. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 98% sequence identity or more thereto.
60. A mucin-binding targeting agent comprising an isolated HC11 BACON-BACON-CBM32 peptide according to SEQ ID NO: 4 or a sequence having 99% sequence identity or more thereto.
61. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 65% sequence identity or more thereto.
62. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 75% sequence identity or more thereto.
63. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 80% sequence identity or more thereto.
64. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 85% sequence identity or more thereto.
65. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 90% sequence identity or more thereto.
66. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 95% sequence identity or more thereto.
67. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 96% sequence identity or more thereto.
68. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 97% sequence identity or more thereto.
69. A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 98% sequence identity or more thereto.
70 A mucin-binding targeting agent comprising an isolated HC12 BACTEROIDES THETAIOTAOMICRON peptide according to SEQ ID NO: 5 or a sequence having 99% sequence identity or more thereto.
71. The mucin-binding targeting agent according to items 1 to 70 wherein the peptide binds to one or more mucins of mammalian origin.
72. The mucin-binding targeting agent according to any one of items 1 to 71 wherein the peptide binds to one or more mucins of human origin.
73. The mucin-binding targeting agent according to any one of items 1 to 72 wherein the peptide is catalytically inactive against mucins.
74. The mucin-binding targeting agent according to any one of items 1 to 73 wherein the mucin to which the mucin-targeting agent binds is one or more of MUC2, MUC5AC, MUC5B, and MUC21.
75. The mucin-binding targeting agent according to any one of items 1 to 74 further comprising a binding moiety selected from the group comprising a peptide linker, an ester, a lipid anchor, avidin, streptavidin, and biotin.
76. The mucin-binding targeting agent according to item 75 wherein the binding moiety is a lipid anchor.
77. The mucin-binding targeting agent according to item 75 wherein the binding moiety is peptide linker.
78. The mucin-binding targeting agent according to item 75 wherein the binding moiety is an ester.
79. The mucin-binding targeting agent according to any one of items 1 to 78 further comprising a payload.
80. The mucin-binding targeting agent according to item 79 wherein the payload is attached to the agent via the binding moiety.
81. The mucin-binding targeting agent according to any one of items 79 or 80 wherein the payload is covalently attached to the binding moiety.
82. The mucin-binding targeting agent according to any one of items 79 to 81, wherein the payload is selected from the group comprising a therapeutic agent, a detectable marker, nanoparticle, a liposome, a vesicle and a stain.
83. The mucin-binding targeting agent according to item 82, wherein the payload is a therapeutic agent selected from the group comprising radioisotopes, enzymes, antibodies, receptors, RNA, DNA, proteins, therapeutic peptides, and oligonucleotides.
84. The mucin-binding targeting agent according to item 82 wherein the payload is a therapeutic peptide.
85. The mucin-binding targeting agent according to any of the preceding items for use as a medicament.
86. A composition comprising the mucin-binding targeting agent according to any of the preceding items.
87. The composition according to item 86, wherein the composition is a pharmaceutical dosage form further comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable carrier.
88. The composition according to item 87 wherein then composition further comprises a shell and/or an enteric coating.
89. The mucin-binding targeting agent according to any of the preceding items for use in the treatment of a disease, illness, or disorder in a subject, wherein the disease, illness, or disorder is selected from the group of from the group of inflammatory, immunological, endocrine, or metabolic disorders such as obesity or may be neurological, psychological or psychiatric or mood disorders, or disorders of the nervous system, or sexual disorders including reproductive disorders and disorders of the genital system, neoplastic disorders such as cancers, disorders involving dysfunction of mucous tissue or dysfunction of epithelial tissue, including disorders, diseases, and illnesses of the gastrointestinal tract, nasal disorders, disorders and diseases of the eye.
90. The mucin-binding targeting agent according to any of the preceding items, wherein the agent is for oral, rectal, vaginal, buccal, ocular, nasal, or inhalation administration.
91. A method of delivery of a payload to a tissue in a subject, said tissue expressing one or more of MUC2, MUC5AC, MUC5B, and MUC21, said method comprising administering to the subject a pharmaceutical composition comprising a mucin-binding targeting agent comprising an isolated peptide according to any one of SEQ ID NO: 1 to 5 or a sequence having 65% or more, such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more thereto and a payload bound to said polypeptide
92. The method according to item 91, wherein the tissue is located in the intestinal tract.
93. A method of preparing a mucin-binding targeting agent, the method comprising the step of providing an isolated peptide according to any one of SEQ ID NO: 1 to 5, or a sequence having 65% or more, such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more thereto.
94. The method according to item 93 further comprising the step of providing a binding moiety and linking the binding moiety and the polypeptide.
95. The method according to item 93 or 94 wherein the peptide and/or the binding moiety is/are produced recombinantly.
96. The method according to any one of items 93 to 95, further comprising a step of attaching a payload to the mucin-binding targeting agent.
97. A mucin-binding targeting agent comprising an isolated mucin-binding peptide sequence that binds an O-glycosylated mucin motif comprised of 5 or more consecutive O-glycans, and which targeting agent do not bind to non-glycosylated mucins independent of the O-glycan structures attached.
98. The mucin-binding targeting agent according to item 97 further comprising a binding moiety.
99. The mucin-binding targeting agent according to item 98 further wherein the binding moiety is selected from the group comprising a peptide linker, an ester, a lipid anchor, avidin, streptavidin, and biotin.
100. The mucin-binding targeting agent according to any one of items 97 to 99 further comprising a payload.
101. The mucin-binding targeting agent according to item 100 wherein the payload is selected from the group comprising a therapeutic agent, a detectable marker, nanoparticle, a liposome, a vesicle and a stain.
102. The mucin-binding targeting agent according to item 101 wherein the payload is a therapeutic agent.
103. A DNA sequence encoding any one of the peptides according to SEQ ID NO: 1 to 5 or a sequence having 65% or more, such as 70% or more, such as 80% or more, such as 85% or more, such as 90 percent or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such 99% or more, such as 99.5% or more sequence identity thereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21177857.6 | Jun 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/065169 | 6/3/2022 | WO |
