Patent Application
20230054093
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled RpI-9013-177_ST25.bd, 52,431 bytes in size, generated on Sep. 6, 2022, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
The invention provides molecules for use in compositions, medicaments and methods for the treatment or prevention of lung inflammatory diseases, pneumonia and/or bronchitis.
Inflammation and exaggerated cytokine/chemokine responses within the lung can lead to a number of damaging pathologies. For example, infection can result in inflammation which causes tissue damage and other complications. Pneumonia and bronchitis are two such complications which can occur as a consequence of viral, bacterial and/or fungal infection. Pneumonia and bronchitis can also occur as a result of ventilation within the intensive care setting and inhalation of toxic substances and chemicals.
In the age of antibiotic resistance there is a need for alternate medicaments, compositions and methods which can be used to prevent or treat lung inflammatory diseases
The present disclosure is based on the finding that molecules with affinity for (or an ability to bind to) sialic acid (and in particular sialoglycoconjugates) on cell surfaces (these including sialic acid containing glycoproteins and cell surface sialic acid receptors), find utility in the treatment and/or prevention of inflammatory diseases and/or conditions with an inflammatory aeitiology. Diseases and/or conditions of this type may include, for example, those diseases and/or conditions characterised by inflammation occurring as a consequence of a cascade of cytokines.
The various molecules described herein may be for use in methods of treating or preventing inflammation that may occur in and around the tissues and/or structures of the lung.
Inflammation in the lung may occur as a result of an infection. As such, the molecules of this disclosure may be used to treat or prevent inflammation that occurs as a result of a microbial, for example, viral, bacterial and/or fungal infection in the lung. One of skill will appreciate that infections of this type can induce potentially harmful cytokine and/or chemokine cascades that lead cell influx (swelling) and tissue damage.
An infection of the lung may lead to a form of bronchitis, bronchiolitis or pneumonia. Accordingly, diseases and/or conditions which may be treated or prevented using the various sialic acid binding molecules described herein may include, for example those selected from the group consisting of:
More specifically, the molecules described herein may be used to treat or prevent one or more of the following types or forms of pneumonia:
Another category of pneumonia which can be treated or prevented by a molecule described herein is “aspiration pneumonia”—this being a pneumonia caused by breathing in vomit, a foreign object or particle and/or a harmful or toxic substance, such as smoke or a chemical.
Other forms of pneumonia which can be treated using a molecule described herein may include, for example those pneumonias which occur in subjects within the hospital or care facility setting, often while said subject is being treated for another, unrelated condition. For example, subjects (or patients) in intensive care on breathing machines are particularly at risk of developing ventilator-associated pneumonia. Accordingly, a molecule of this invention may be used, perhaps prophylactically, to ensure that the risk of pneumonia is reduced in subjects who are vulnerable/predisposed to pneumonia (this would include patients or subjects with underlying health issues, intensive care patients (particularly those who are being ventilated) and/or immunocompromised patients).
Other diseases and/or conditions, including those without a microbial aeitiology, may also result in some level of inflammation in the lung. Diseases and/or conditions of this type can be treated and/or prevented using any of the molecules described herein and may include (for example) one or more of the diseases listed below:
For convenience, all of the diseases and conditions described herein shall be referred to under the term “lung inflammatory disease”.
The present disclosure provides a sialic acid binding molecule for use in the treatment and/or prevention of lung inflammatory disease.
Further provided is the use of a sialic acid binding molecule in the manufacture of a medicament for use in the treatment and/or prevention of lung inflammatory disease.
The disclosure also provides a method of treating or preventing a lung inflammatory disease, said method comprising administering a subject in need thereof a therapeutically effective amount of a sialic acid binding molecule.
The disclosure also provides sialic acid binding molecules and medicaments and methods comprising sialic acid binding molecules, for use in methods of treating or preventing a lung inflammatory disease.
Throughout this specification, the terms “comprise”, “comprising” and/or “comprises” is/are used to denote aspects and embodiments of this invention that “comprise” a particular feature or features. It should be understood that this/these terms may also encompass aspects and/or embodiments which “consist essentially of” or “consist of” the relevant feature or features.
Without wishing to be bound by theory, it is suggested that when administered to a subject, a sialic acid binding molecule of this disclosure can suppress, inhibit or reduce a cytokine response within the lung and/or suppress, inhibit or reduce the influx of cells, including, immune cells, into the lung tissue. Combined, these actions result in a reduced inflammatory response within the lung and also reduce any damage typically associated with exaggerated cytokine/chemokine cascades.
While it may have been established that certain sialic acid binding proteins can be used to prime or modulate the immune system by increasing the levels of certain cytokines (including various proinflammatory cytokines), and that a primed or modulated immune response may impact on the pathology of a whole host of different pathogens (including those that do not bind or which do not primarily bind sialic acid during pathogenesis), one of skill would still not have been led to the finding that the sialic acid binding molecules described herein inhibit cytokine responses and cell migration and can therefore be used to treat or prevent diseases or conditions which are caused or contributed to by activated cytokines and increased cell migration.
Further, for prior art disclosures where sialic acid binding molecules have been used as agents capable of blocking the binding of pathogens to cell surface sialic acid/sialoglycoconjugates, the utility of the sialic acid binding molecule is rooted in the fact that both the sialic acid binding molecule and the pathogen bind sialic acid; this is not the case here. Many of the pathogens or clinical situations that lead to lung inflammatory diseases of the type described herein, do not concern or involve binding between the pathogen and sialic acid. As stated, the present disclosure is based on the unexpected finding that certain sialic acid binding molecules act to inhibit specific cytokines (including proinflammatory cytokines) and cell migration.
The findings reported in this disclosure have important implications for the formulation and administration of molecules with sialic acid binding affinity and for the subsequent use of these formulations in the treatment and/or prevention of lung inflammatory diseases.
For example, the finding that the sialic acid binding molecules described herein can be used to treat or prevent a lung inflammatory disease allows for the use or preparation of the disclosed sialic acid binding molecules as formulations suitable for mucosal, intranasal or inhalation administration.
Accordingly, the disclosure provides a composition for mucosal administration, said composition comprising a sialic acid binding molecule for use in the treatment and/or prevention of lung inflammatory diseases.
It should be noted that the term “mucosal administration” embraces compositions that have been formulated for administration to any mucosal surface, including, for example, respiratory surfaces, nasal passages and the like. The term also embraces compositions formulated for administration by inhalation. Compositions suitable (or formulated) for mucosal administration may include compositions, which are intended to be administered intranasally (or nasally).
Thus a composition for mucosal administration may be formulated with excipients, diluents and/or buffers which are suitable for use in any type of mucosal administration as described above.
Compositions for mucosal (for example intranasal) administration may comprise solutions of the sialic acid binding molecule(s) to be administered and/or particles (comprising the same) for aerosol dispersion or dispensed in drinking water. When dispensed such compositions should desirably have a particle diameter in the range 10 to 200 microns to enable retention in, for example, the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable compositions include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension. A composition for mucosal administration may be provided in the form of a liquid spray.
Importantly, this disclosure provides sialic acid binding molecules for prophylactic use. Specifically, the sialic acid molecules described herein may be used prophylactically in order to prevent a lung inflammatory diseases in subjects.
Accordingly, the present disclosure provides a sialic acid binding molecule for use in the prevention a lung inflammatory disease. Also provided is the use of a sialic acid binding molecule in the manufacture of a medicament for use in the prevention of a lung inflammatory disease. A method of prophylaxis or preventing a lung inflammatory disease may comprise administering a subject in need thereof a therapeutically effective amount of a sialic acid binding molecule. In all cases, the sialic acid binding molecule may be:
Additionally, in all cases, the lung inflammatory disease may be a pneumonia, bronchiolitis and/or bronchitis.
As used herein and in any method of prophylaxis or method or sialic acid binding molecule for use in preventing a lung inflammatory disease, the term “subject” may extend to any subject predisposed, susceptible or at risk of developing a lung inflammatory disease, including pneumonia, bronchiolitis and/or bronchitis. The subject may be a neonate, an infant or a child. The subject may be an adult or elderly. The subject may be an immunocompromised subject. The subject may have one or more underlying or chronic health problems—in particular, problems affecting the respiratory tract and/or respiration. For example, the subject may suffer from asthma. The subject may have a viral, bacterial and/or fungal infection; that infection may reside within the lung of the subject. The subject may be an intensive care patient.
Throughout this specification, the term “sialic acid binding molecule” and this embraces any useful sialic acid binding molecule. Useful sialic acid binding molecules may take any form and/or belong to any class of molecule or compound (for example they may be proteins, peptides, carbohydrates, antibodies and the like) and the term “sialic acid” embraces all forms of N- or O-substituted neuraminic acid and includes all synthetic, naturally occurring and/or modified forms thereof. Sialic acids may be found as components of cell surface molecules, glycoproteins and glycolipids. Most often, sialic acids are present at the end (terminal regions) of sugar chains connected to cell membranes and/or proteins. For example, some cells of the human upper respiratory tract comprise α-2,6-linked sialic acid receptors and other cells of the upper and lower respiratory tracts comprise α-2,3-linked sialic acid receptors. The sialic acid family encompasses a number (approximately 50) of derivatives that may result from acetylation, glycolylation, lactonisation and methylation at C4, C5, C7, C8 and C9. All such derivatives are to be embraced by the term “sialic acid”.
Furthermore, sialic acids are found linked α(2,3) or α(2,6) to Gal and GaINAc or α(2,8) or α(2,9) to another sialic acid. Accordingly, it is important to understand that while the term “sialic acid” is used throughout this specification, it encompasses all derivatives, analogues or variants (either naturally occurring or synthetically generated) thereof as well as monomers, dimers, trimers, oligomers, polymers or concatamers comprising the same.
Thus, a sialic acid binding molecule of this disclosure (and for use as described herein) comprises a moiety which exhibits an affinity for sialic acid—which may include all forms of sialic acid described above and any form of sialic acid present on the surface of a cell (perhaps as part of a cell surface receptor), for example a mammalian cell. These various forms of sialic acid may be collectively referred to as “sialic acid moieties”.
The sialic acid binding molecules of this disclosure exhibit an affinity for sialic acid and as such they may bind/couple to and/or associate with one or more sialic acid moieties. Thus, the term “sialic acid binding molecule” may further encompass fragments of whole sialic acid binding molecules which retain an ability to bind to or otherwise couple or associate with a sialic acid moiety.
Sialic acid binding molecules, including those for the uses, compositions and methods described herein, may comprise a single sialic acid binding molecule (a monomeric or monovalent molecule, for example) or, alternatively, two or more sialic acid binding molecules—which two or more molecules may be the same or different—a polymeric or multivalent molecule, for example.
A sialic acid binding molecule, including those for uses, compositions and methods described herein may comprise, consist essentially of or consist of, one or more of the sialic acid binding molecules known as “carbohydrate binding modules” (CBMs). CBMs suitable for use exhibit an affinity for sialic acid. CBMs are classified into families and CBMs classed as members of the family 40 CBMs (CBM40) may be useful. The family 40 CBMs embrace molecules of approximately 200 residues and are often found at the N-terminus of GH33 sialidases. They may also be found inserted in the β-propeller of GH33 sialidases.
The disclosure may embrace the use of molecules, for example, larger molecules, which comprise a sialic acid binding component. As stated, that sialic acid binding component (i.e. the sialic acid binding molecule) may itself comprise (consist of or consist essentially of) a CBM, for example, a CBM40. By way of (non-limiting) example, the molecules (e.g. the sialic acid binding molecules) of this disclosure may not only exhibit an ability to bind sialic acid, but may also have one or more other functions. For example, the molecules may have enzymatic activity. For example, a useful molecule may comprise a CBM (as described herein) and exhibit some sialidase activity.
A useful sialic acid binding molecule may be a fusion protein comprising an enzymatic portion and a sialic acid binding portion—wherein the sialic acid binding portion comprises a sialic acid binding molecule of this disclosure. In such cases, the enzymatic portion may be fused to the sialic acid binding portion. As stated, the enzymatic portion of any useful fusion protein may comprise (or have, or exhibit) sialidase activity.
The sialic acid binding protein or CBM for the various uses, methods and compositions described herein, may not be provided as part of, or comprised within, a molecule (for example a fusion protein) with enzymatic (for example sialidase) activity. Additionally or alternatively, the sialic acid binding molecule may not (i) bind heparin or heparin sulfate and/or (ii) comprise the GAG-binding domain of a protein that binds heparin or heparin sulfate moieties.
As such, the present disclosure provides a CBM or CBM40 for use in the treatment and/or prevention of the lung inflammatory disease disclosed herein.
Further provided is the use of a CBM or CBM40 in the manufacture of a medicament for use in the treatment and/or prevention of the lung inflammatory diseases disclosed herein.
The disclosure also provides a method of treating or preventing a lung inflammatory disease of this disclosure, said method comprising administering a subject in need thereof a therapeutically effective amount of a CBM or CBM40.
The disclosure also provides sialic acid binding molecules and medicaments and methods comprising a CBM or CBM40 for use in methods of treating or preventing a lung inflammatory disease such as, for example pneumonia, bronchiolitis and/or bronchitis.
The disclosure provides a composition for mucosal administration, said composition comprising a CBM and/or CBM40 for use in the treatment and/or prevention of the lung inflammatory diseases of this disclosure. As stated, a composition for mucosal administration may be formulated with excipients, diluents and/or buffers which are suitable for use in any type of mucosal administration, including, for example intranasal administration.
The disclosure may further provide a CBM or CBM40 for prophylactic use. Specifically, CBM or CBM40 described herein may be used prophylactically in order to prevent infection lung inflammatory disease, including, for example pneumonia, bronchiolitis and/or bronchitis.
Exemplary carbohydrate binding modules (CBMs) may comprise the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM: a CBM40) and/or the equivalent (or homologous) domain from Streptococcus pneumoniae NanA sialidase (SpCBM: also a CBM40). Of course, similar or homologous sialic acid binding modules present in other organisms are to be encompassed within the scope of the term “CBM”.
An exemplary Vibrio cholerae NanH sialidase amino acid sequence is deposited under accession umber A5F7A4 and is reproduced below as SEQ ID NO: 1 (781 amino acids).
The CBM region of SEQ ID NO: 1 is from amino acid residue 25 to 216—this sequence may be SEQ ID NO: 2.
An exemplary Streptococcus pneumoniae NanA sialidase amino acid sequence has been deposited under accession number P62575 and is reproduced below as SEQ ID NO: 3 (1035 amino acids).
The CBM region of SEQ ID NO: 3 is from amino acid residue 121 to 305—this sequence may be SEQ ID NO: 4.
Thus, CBMs for use as sialic acid binding molecules in the various aspects and embodiments of this disclosure may comprise a protein or peptide having the sequence of SEQ ID NO: 1, 2, 3 or 4 or a sequence or fragment derived therefrom. Sequences or fragments derived from any of SEQ ID NOS: 1, 2, 3 or 4 may themselves provide or encode a molecule with an ability to bind sialic acid (in other words a sialic acid binding molecule encoding portion of fragment of SEQ ID NOS: 1, 2, 3 or 4).
A sialic acid binding molecule for use may comprise an amino acid sequence which is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any of the sequences provided by SEQ ID NOS: 1, 2, 3 or 4.
Further, a sialic acid binding molecule for use may comprise from about residue 1, 5, 10, 15, 25 or 30 (i.e. from 1-30 or from any amino acid residue there between) to about residue 150, 175, 200, 210, 216, 220-781 (to any residue from 150 to 781 including any residue therebetween) of the V. cholerae sialidase molecule of SEQ ID NOS: 1 and 2. For example a sialic acid binding molecule for use may comprise a peptide having a sequence corresponding to residue 25 to about residue 216 of SEQ ID NO: 1 above.
A sialic acid binding molecule for use may comprise from about residue 80, 90, 100, 110, 120, 121 to 130 (i.e. from any of about residues 80 to 130 including any residue therebetween) to about residue 250, 275, 300, 305, 310, 320-1035 (i.e. to any residue from about 250-1035 including to about any residue therebetween) of the S. pneumoniae sialidase molecule of SEQ ID NOS: 3 and 4. For example, a sialic acid binding molecule for use may comprise a peptide having a sequence corresponding to residue 121 to about residue 305 of SEQ ID NO: 3 above.
A sialic acid binding molecule for use may comprise one or more CBMs. For example, suitable sialic acid binding molecules may comprise single CBM—for example a single sialic acid binding molecule derived from VcCBM (an exemplary VcCBM sequence being disclosed above as SEQ ID NOS: 1 and 2) or a single sialic acid binding molecule derived from SpCBM (an exemplary SpCBM sequence being disclosed above as SEQ ID NOS: 3 and 4). Alternatively, a sialic acid binding molecule for use may comprise a plurality or multiple (i.e. two or more) CBMs. Sialic acid binding molecules which comprise a plurality of CBMs may be termed “multivalent sialic acid binding molecules” or “multivalent CBMs”. A multivalent CBM may, for example, comprise two or more (for example three, four, five or six) sialic acid binding molecules derived from VcCBM or two or more sialic acid binding molecules derived from SpCBM. A multivalent CBM may comprise a mixture of different CBMs, for example one or more sialic acid binding molecules derived from VcCBMs with one or more sialic acid binding molecules derived from SpCBMs.
The sialic acid binding molecules for use may further comprise an oligomerisation domain. Suitable oligomerisation domains may exhibit an ability to self-associate to form multimer structures, for example trimers. An oligomerisation domain for use may comprise any molecule with the above mentioned oligomerisation properties or any functional fragment thereof. For example, one or more (for example two) sialic acid binding molecules (for example sialic acid binding molecules derived from the CBMs described herein) may be bound, coupled or fused to an oligomerisation domain—the resulting sialic acid binding molecule::oligomerisation domain “fusion” may then be used (with one or more other such “fusions”) as a molecule for modulating cell growth and/or activity and/or for treating or preventing any of the diseases and/or conditions disclosed herein.
Suitable oligomerisation domains may be derived from, for example, Pseudomonas aeruginosa pseudaminidase. An exemplary Pseudomonas aeruginosa pseudaminidase sequence amino acid sequence has been deposited under accession number PA0579 and is reproduced below as SEQ ID NO: 5 (438 amino acids).
The oligomerisation domain of SEQ ID NO: 5 is from amino acid residue 333 to 438—this sequence may be SEQ ID NO: 6.
Thus an oligomerisation domain for use may comprise an amino acid sequence which is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequences provided by SEQ ID NOS: 5 or 6.
Further, an oligomerisation domain for use may comprise from about residue 250, 275, 300, 310, 320, 333, 340 to 350 (i.e. from about residue 250 to about residue 350 including from about any residue therebetween) to about residue 400, 410, 420, 430 or 438 (i.e. to about any residue from about residue 400 residue 438 including to about any residue therebetween) of the P. aeruginosa pseudaminidase trimerisation domain (PaTD) provided by SEQ ID NO: 5. For example, a useful sialic acid binding molecule may exploit an oligomerisation domain comprising residues 333 to 438 of SEQ ID NO: 6.
Other sialic acid binding domains may comprise two or more SpCBMs optionally fused, bound or conjugated to an oligomerisation domain (such as a PaTD or an oligomerisation fragment thereof). Sialic acid binding molecules may comprise, consist or consist essentially of two fused (or bound) SpCBMs which are in turn fused to an oligomerisation domain (see, for example, molecule Sp2CBMTD shown in
This disclosure refers to useful sialic acid binding molecules which are “derived” from the various sialic acid binding molecules described herein (including, for example, the various CBMs of this disclosure”. These “derived” (and useful) molecules may comprise sialic acid binding molecules which represent modified forms of any of the molecules described herein, including modified forms of the disclosed CBM sequences.
It should be understood that the term “modified” embraces molecules which contain one or more mutations relative to a reference sequence.
In the context of this disclosure, a “reference sequence” may be any wild type sequence encoding or providing a sialic acid binding molecule, for example a wild type CBM sequence. For example, a reference sequence may comprise, consist essentially of or consist of a wild type family 40 CBM sequence, e.g. the wild type CBM sequences from Vibrio cholerae NanH sialidase or Streptococcus pneumoniae NanA sialidase (it should be appreciated that similar or homologous CBMs (including CBM40s) present in other organisms are to be encompassed within the scope of the term “CBM” and/or as CBM reference sequences). A reference sequence from which a useful sialic acid binding molecule may be derived (including useful multivalent CBMs as described herein) may comprise any of the specific sequences described herein (for example SEQ ID NO: 1, 2, 3, 4 and 5.
For example, a modified CBM sequence for use may be derived from a specific or particular wild type CBM. A useful modified CBM sequence may comprise a wild type CBM sequence which includes one or more mutations.
The one or more mutation(s) may be functional. The mutations may, for example, alter the overall primary sequence of a CBM for use, but may not (substantially) alter the properties of the CBM—thus, while the sequence of a modified CBM may be different from the wild-type sequence from which it is derived, the overall function of the modified CBM is (substantially) identical to that of the wild-type CBM. Alternatively, the one or more mutation(s) may individually (and/or independently) or collectively (for example synergistically) modulate (improve or suppress/inhibit) one or more of the physiological, biological immunological and/or pharmacological properties characteristic of a wild type CBM (for example the wild type CBM from which the modified CBM is derived). In particular, the one or more mutations may:
A “mutation” may include any alteration to a wild-type CBM molecule. For example, the term “mutation” may embrace, for example:
Thus, a useful modified CBM (i.e. a CBM for use in the medical uses and methods described herein) may comprise one or more of the mutations described herein.
By way of non-limiting example, the following represent individual units (referred to as “HEX” units) which may be used to make hexameric sialic acid binding molecules which have a particular application in the various compositions, medicaments, methods and uses described herein (for example for use in methods of treating or preventing lung inflammatory diseases, including pneumonia and/or bronchitis). In each case, the HEX unit comprises two modified CBMs (denoted CBM1 and CBM2), which modified CBMs are derived from the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM: a CBM40 family member). The specific mutations introduced to each modified CBM are identified in parenthesis. It should be noted that a “- - - -” symbol indicates an amino acid linker (linking one CBM to another or a CBM to an oligomerisation domain). As such, a hexameric (‘HEX’) sialic acid binding molecule may be made up of several (for example 3) HEX units. The oligomerisation domain (denoted “TD”) conjugates the units together as a trimer. While any given hexamer may comprise identical copies of the units described above (and below under the headings HEX1 unit, HEX2 unit, HEX3 unit, HEX4 unit, HEX5 unit, HEX6 unit and HEX17 unit), one of skill will appreciate that further options are available. For example, a HEX unit may be made up of two CBMs, each having different mutations (the mutations being one or more selected from the options detailed herein).
It will be noted that HEX6 and HEX17 are identical except for the additional A162P mutation. This proline mutation (a substitution for the wild type Alanine at residue 162) has been shown to improve thermostability (the single CBM Tm by 3-4° C.). Further information regarding the use of proline mutations may be derived from Fu 2009, ‘Increasing protein stability by improving beta-turns’ (DOI 10.1002/prot.22509) which describes the general approach. The proline mutation does not affect (increase or decrease) the predicted immunogenicity of the CBM molecule, is not located near the other mutations, the N- or C-termini or the ligand binding site. Rather unexpectedly, beyond the modest improvement in thermostability, it was noted that the A162P mutation yields a hexameric CBM (i.e. a molecule comprising 3×HEX17 units) exhibiting a marked improvement in in vivo experiments—in particular in comparison to those same experiments conducted using a hexameric molecule comprising 3×Hex6 units. For example, the modified molecules (in particular a molecule comprising 3×HEX17 units) exhibit modulation over pro-inflammatory cytokines, including for example IL-8. Indeed the modulatory effect (specifically an inhibitory effect) on the production of IL-8 by a molecule comprising 3×HEX17 units, was improved over other tested modified molecules.
Relative to the amino acid sequences of Sp2CBMTD (aka “SpOrig” SEQ ID NO: 7) the amino acid sequence of the HEX6 (SEQ ID NO: 8) and HEX 17 (SEQ ID NO: 9) molecules is:
The disclosed molecules, including those for use in the described compositions, medicaments and methods may be generated using PCR-based cloning techniques and a suitable method for the generation of multivalent molecules of this type is described in, for example, Connaris et al, 2009 (Enhancing the Receptor Affinity of the Sialic Acid-Binding Domain of Vibrio cholerae Sialidase through Multivalency; J. Biol. Chem; Vol. 284(11); pp 7339-7351). For example, multivalent CBM molecules, including the likes of HEX17 and/or HEX17 variants may be prepared as constructs comprising multiple sialic acid binding molecules (for example modified CBMs) linked by amino acid/peptide linkers.
As stated, each CBM (for example modified CBM) may be linked to another by, for example, peptides comprising 5, 10 or 15 amino acids. By way of example any one or more of the following peptides may be used to link two or more CBMs (including the described modified CBMs) to produce a multivalent CBM:
where “X” is any amino acid.
Thus an exemplary sialic acid binding molecule (for example a HEX17 unit) may take the following form:
This schematic shall be referred to hereinafter as General Formula 1.
Thus, a HEX17 unit may conform to General Formula 1 as set out above, comprising two modified CBMs (“Mod CBM 1” and “Mod CBM 2”) and a trimerisation domain, TD wherein Peptide Linkers A and/or B are selected from the linker options presented above as (i), (ii) and/or (iii).
It should be noted that the term “HEX17” embraces not only the complete HEX17 sequence described above but also functional (for example, sialic acid binding and/or anti-inflammatory) fragments derived therefrom. Indeed, each of the “Mod CBM” units shown in General Formula 1 may be the HEX17 units shown above.
In view of the above, a HEX17 molecule for use in the various aspects of this disclosure comprises three HEX17 units bound together via the trimerisation domain.
Accordingly, the various uses, compositions, sialic acid binding molecules for use, methods and medicaments described herein may exploit sialic acid binding molecules which comprise, consist of or consist essentially of sialic acid binding molecules selected from the group consisting of:
For the avoidance of doubt, the term “HEX17” includes sialic acid binding molecules having a sequence corresponding to that provided by SEQ ID NO: 9 or which exhibits some level of sequence identity thereto (for example, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 9). Sialic acid molecules with sequences exhibiting some level of identity to SEQ ID NO: 9 (which level of identity is selected from the % identity values disclosed above) may be referred to as “HEX17 variants”. HEX17 variants may be functional in that they bind to sialic acid and/or are anti-inflammatory (i.e. they inhibit the production or expression of certain proinflammatory cytokines).
As such, the present disclosure provides:
The disclosure also provides the use of HEX17 and/or a HEX17 variant in the manufacture of a medicament for use in the treatment and/or prevention of a lung inflammatory disease, pneumonia, bronchiolitis and/or bronchitis.
The disclosure also relates to a method of treating or preventing a lung inflammatory disease, pneumonia, bronchiolitis and/or bronchitis, said method comprising the steps of administering to a subject in need thereof, a therapeutically effective amount of HEX17 and/or a HEX17 variant.
For completeness, it should be noted that Vc2CBM comprises, consists essentially of or consists of two Vibrio cholerae NanH sialidase CBM units linked, bound or conjugated together. An exemplary Vc2CBM sequence may comprise, consist essentially of or consist of:
Additionally, Vc4CBM comprises, consists essentially of or consists of four Vibrio cholerae NanH sialidase CBM units linked, bound or conjugated together. An exemplary Vc4CBM sequence may comprise, consist essentially of or consist of the following sequence:
Also, Sp2CBM comprises, consists essentially of or consists of two Streptococcus pneumoniae NanA sialidase units linked, bound or conjugated together. An exemplary Sp2CBM sequence may comprise, consist essentially of, or consist of two copies of the following sequence:
The two copies of the abovementioned sequence may be joined via any one of the peptide linker sequences described herein. For example, a Sp2CBM sequence may comprise, consist essentially of, or consist of
xx
]
[
xxxxxxxxxxxxxxx
]GSMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAP
Wherein [xxxxx], [xxxxxxxxxx] and [xxxxxxxxxxxxxxx]—represent the choice of linker peptide sequences as outlined below. Both CBM sequences would be joined by one of the 5, 10 or 15 amino acid linker sequences described herein.
Vc2CBM and Vc4CBM may be described as tandem-repeat multivalent proteins based on the Family 40 sialic acid binding domain (CBM) of the nanH gene encoding the sialidase from V. cholerae. Sp2CBM may be described as a tandem-repeat multivalent protein based on the family 40 sialic acid binding domain (CBM) of the nanA gene encoding the sialidase from S. pneumoniae.
Further, it should be noted that the various uses, methods and medicaments described herein may exploit one or more of the sialic acid binding molecules described herein. For example, a sialic acid binding molecule comprising HEX17 (i.e. a sequence according to SEQ ID NO: 9) or a HEX17 variant may be administered to a subject together, concurrently or separately with another sialic acid binding molecule or and/or other therapeutic moiety or adjuvant.
The present disclosure provides compositions for the various uses, medicaments compositions, and methods described herein. As such, any of the useful sialic acid binding molecule(s) (for example the HEX17 or HEX17 variants) described herein may be formulated for subsequent use. For example, a sialic acid binding molecule (or molecules) may be formulated as therapeutic or pharmaceutical compositions. The various compositions may comprise one or more of the sialic acid binding molecules described herein and any given treatment may require the administration (together, concurrently or separately) of one or more of these compositions.
Pharmaceutical compositions according to the present invention, in particular those formulations for mucosal or intranasal administration may be prepared conventionally, comprising substances that are customarily used in pharmaceuticals and as described in, for example, Remington's The Sciences and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press 2012) and/or Handbook of Pharmaceutical Excipients, 7th edition (compiled by Rowe et al, Pharmaceutical Press, 2012)—the entire content of all of these documents and references being incorporated by reference.
Any suitable amount of a sialic acid binding molecule (for example, the HEX17 molecules described herein) may be used. For example, whether a composition comprising a sialic acid binding molecule (for example HEX17) is to be administered intravenously or mucosally (for example, intranasally) the dose of sialic acid binding molecule may comprise anywhere between about 0.1 μg and about 1000 μg. For example, a dose of about (for example +/−0.5 μg) 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg or 950 μg of the sialic acid binding molecule may be used. These amounts may be provided in any suitable volume of excipient, diluent or buffer. For example, the amount of sialic acid binding molecule may be provided in anywhere between about 1 μl to about 5 ml of excipient, diluent or buffer. For example, the required amount of sialic acid binding molecule may be combined (or formulated) with about 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl, 75 μl, 80 μl, 85 μl, 90 μl, 95 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 ml, 2 ml, 3 ml or 4 ml. Concentrations of 0.1-1 mg (sialic acid binding protein) per ml (excipient, diluent or buffer) may be most useful.
A composition of this disclosure (for example, a composition comprising HEX17 or a HEX17 variant) may be administered (prophylactically) to a subject at regular and/or predetermined times. For example, a composition described herein may be administered to at regular and/or predetermined times both before, during and after the subject enters or encouters a scenario during which they might be vulnerable and/or susceptible to a lung inflammatory disease (such as, for example pneumonia and/or bronchitis). A composition of this disclosure may be administered every day and/or every few days. A composition of this disclosure may be administered multiple times throughout any given day. A composition described herein may be administered over a period of weeks or months or years. The precise administration regimen will depend on the subject, the health of that subject and the period of time that subject is deemed to be at risk of or vulnerable to, a lung inflammatory disease (such as pneumonia or bronchitis).
The present invention will now be described in detail with reference to the following figures which show:
The in silico T-cell epitope screening identified four significant and two borderline immunogenic clusters:
The ProImmune study highlighted two regions of high antigenicity and two regions of moderate antigenicity:
A further in silico tool, the online ProPred server4, was also used. The output of the ProPred server is shown in
To guide the design of mutations that might reduce immunogenicity, ProPred was used to test the effect of changing each residue in these peptides to every alternative residue. Those that gave the greatest reduction in predicted number of allele binders were noted. As the crystal structure of both the SpCBM and TD domains are known, these mutations were also modelled to reduce the likelihood of introducing mutations that would obviously disrupt the protein structure.
Initially, nine single mutations in SpCBM and four single mutations in PaTD were introduced and are listed below (‘Im’ is short for immunogenicity mutant):
The genes encoding WT SpCBM, WT PaTD and the variants Im15 to Im27 were codon optimized for E. coli expression and synthesized by GeneArt. The genes were then cloned in-house into the pHISTEV vector for expression as 6His-tagged proteins.
An initial expression test was performed to assess solubility. The results show that all were expressed, but not all were soluble (
Results of the expression test show that:
The 13 soluble proteins were expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC), followed by TEV digestion to remove the 6His-tag, then reverse IMAC and size exclusion chromatography (SEC).
Ten purified domains (WTSp, Im19, Im20, Im21, Im22, Im23, WTTD, Im24, Im26 and Im27) were further characterized by:
(i) Thermofluor to measure melting temperature (Tm)
(ii) Near UV circular dichroism (CD) to compare tertiary structures to WT
(iii) Dynamic light scattering (DLS) to check oligomeric state in solution
(iv) Surface plasmon resonance (SPR) to measure binding affinity to sialyllactose
(v) Measurement of IL-8 cytokine stimulation
The results are summarized in Table 1.
Im15, Im16, Im17, Im18 are all insoluble or poorly soluble (as stated, this does not necessarily impact on protein utility). These are in the ‘moderately’ antigenic region 167-181 (FYNLFSVSSATKKDE). This region is clearly very sensitive to change.
Earlier results show that M156F, which sits adjacent to L170 (and I286), increases Tm by ˜4° C.
This could therefore be combined with L170T. M156F does not increase predicted immunogenicity.
M185I increases Tm by 5° C., and lies parallel to L170 (
Im19, Im20, Im21 all behave similarly to WT. These are in the ‘highly’ antigenic region 236-250 (KGRVRLYVNGVLSRT).
Im19 (V239A) was chosen over the threonine mutation (Im20, V239T). There is no difference in predicted immunogenicity but Im19 is a closer match to WT Thermofluor Tm and Near UV spectrum. This would be combined with Im21 (V246G).
Im22 (I286A) is broadly similar to WT while Im23 (Y292E) appears to exhibit reduced ligand affinity. This region, 286-294 IRNLTVYNR, was not flagged up by ProImmune but is strongly predicted by ProPred to be immunogenic.
There is some indication that Im22 has lower Tm than WT. This residue is adjacent to M156 so may behave differently if M156F was included.
Im24 (S342D) and Im26 (L348D) show similar characteristics to the WT trimerization domain, but with some suggestion of reduced Tm in Im26. These are in the ‘moderately’ antigenic region 338-352 SDWFSVSSNSLYTLS. The WT sequence was predicted to bind 9 alleles, while Im24 predicts 2 alleles and a Im24/Im26 double mutant predicts 1 allele.
Im27 (R403K) is similar to WT. It is part of the ‘highly’ antigenic region 392-406 GAQVEVGSLNIRLGT. Predicted alleles are reduced from 21 to 3 when this mutation is introduced.
The following mutations were introduced:
i) M156F/L170T
ii) M156F/L170T/M185I: In ProPred, alleles predicted for this region are reduced from 31 in the WT to 19 for this combination.
iii) V239A/V246G: In ProPred, alleles for this region are reduced from 44 to 3.
iv) I286A/Y292E: In ProPred, alleles are reduced from 41 to 1.
v) V239A/V246G/I286A/Y292E combines the previous two doubles.
vi) M156F/L170T/M185I/V239A/V246G/I286A/Y292E combines all the Sp mutations
vii) TD: S342D/L348D/R403K: Predicted alleles are reduced from 9 to 1 for TD peptide 338-352 and alleles for peptide TD peptide 392-406 are reduced from 21 to 3. This triple mutant combines all the TD mutants. They are all surface exposed and distal to the N-terminal end of TD, so would not be expected to interfere with SpCBM in the hexamer form.
The constructs are named Im28 to Im34:
As with the single mutations, the combinations Im28 to Im34 were synthesized by GeneArt and subcloned into pHISTEV for expression analysis. A nickel bead pull-down on the His-tagged soluble extract was also performed (
Genes encoding the hexameric forms (called HEX1 to HEX17) were synthesized by GeneArt:
The hexameric forms were synthesized in two parts to avoid problems associated with synthesising repeat sequences in the tandem CBM copies. The first gene covered the first CBM and the second part encompassed the second CBM plus the TD. These could then be simultaneously cloned into pHISTEV to create the Sp2CBMTD construct that trimerizes upon expression.
The first hexamer, HEX1, contained the mutations L170T/V239A/V246G/I286A/Y292E in the CBMs and S342D/L348D/R403K in the TD.
The solubility data of the individual domains indicated that HEX1 was unlikely to be soluble (again, not necessarily a reflection on the utility of the molecule); a further construct, HEX3, was synthesized. Note that HEX2 contained the same mutations as HEX3, but with the addition of Y292E.
HEX3 was synthesized and subcloned into the pHISTEV vector. Expression was insoluble under all conditions tested (varying temperature, IPTG concentration, cell density at induction, with or without heat shock). The CBM-only domain containing the same three mutations (V239A V246G I286A) is soluble. A double mutant (V239A V246G) behaves very similarly to WT. Therefore, further variants (HEX4, HEX5 and HEX6) were designed and constructed by PCR/ligations, which exclude I286A and contain either one or both of the TD mutations.
During the work on HEX6 a number of other versions were designed containing different combinations of the HEX6 mutations (numbered HEX7 to HEX16; not characterised).
HEX17 contains the HEX6 mutations with an additional A162P mutation. This proline mutation has been shown to increase the single CBM Tm by 3-4° C. The proline mutation is not near the other mutations, the N- or C-termini or the ligand binding site.
The expression, purification and characterization results are shown in Table 2. Based on these results, HEX6 and HEX17 were taken forward. The positions of the HEX17 mutations on the hexamer are shown in
Aim: To Measure the Innate Immune Response of mCBM-Treated Human Lung Epithelial Cells (A549) by Analysing Levels of Inflammatory Mediators Over Time.
Administration of Sp2CBMTD to mammalian cells stimulated a pro-inflammatory response both in vitro and in vivo1,2. To determine whether this was still observed with modified hexameric sialic acid binding molecules, mammalian A549 cells were stimulated by the addition of 10 μg of biologic (Sp2CBMTD (aka SpOrig), HEX6 (i.e. a sialic acid binding molecule comprising 3×HEX6 units) or HEX17 (i.e. a sialic acid binding molecule comprising 3×HEX17 units) and cell culture medium was harvested at specific time-points post administration. The concentrations of inflammatory mediators were measured both by ELISA and a multiplex assay.
Human IL-8 (benchmark cytokine for the study) response using a human 1× Mouse CXCL1/KC Quantikine ELISA Kit (R&D BioSystems). The concentration levels of IL-8 from stimulated A549 cells are shown in
Inflammatory mediator response using a Human Cytokine 12-plex Assay (Bio-Plex Pro™, Bio-Rad).
Supplied by Pneumagen. Quantity: 10×1 mL aliquots of phosphate buffered saline (10×) diluted to 1× using MilliQ water pH 7.2. Manufacturer: Life Technologies. Product Number: 70013-016. Storage: Room temperature or 20° C.
Supplied by Pneumagen (Batch Number: 20190410A; Certificate Number: PGN0030/290419). Quantity: 11×100 μL aliquots of protein at a concentration of 10 mg/mL storage: −20° C.
Non-fasted mice (female BALB/c, 17-20 g) were weighed, individually identified on the tail with a permanent marker and then infected intranasally with RSV or virus diluent (DMEM, 2% v/v FCS, 12.5% w/v Sucrose) under isoflurane (5% in O2) anesthesia. The A2 strain of RSV (50 μL of 5×106 PFU) was instilled into each nostril in a drop-wise fashion, alternating between the two until a volume of 50 μL has been delivered. At the outset of the study, the A2 strain of RSV was back-titrated to determine viability of the virus.
Throughout the study the test vehicle and test agent were formulated as detailed below.
On each day of dosing, an aliquot (1 mL vial) of the test vehicle was diluted from 10× to 1× to a volume of 10 mL using endotoxin-free water. 1×PBS was then used for Neumifil dilution and also for dosing the vehicle control group.
On each day of dosing, an aliquot of Neumifil (100 μL per vial at 10 mg/mL) was thawed and transferred into a new sterile 0.5 mL Eppendorf tube and centrifuge at 13,000 rpm for 5 min. The supernatant was transferred into new sterile 1.5 mL Eppendorf tubes and diluted to a volume of 400 μL using 1×PBS to formulate a concentration of 2.5 mg/mL (equivalent to 100 μg/40 μL). Neumifil tubes were clearly labelled and kept at RT. Prior to dosing, the formulation contents were gently mixed using a pipette, avoiding bubbles.
A new vial of Neumifil or vehicle was used for each group and day of dosing. Remaining formulations (both diluted and non-diluted Neumifil) were stored at −20° C. and shipped back to Pneumagen at the end of the study.
Test agent (Neumifil) or vehicle (PBS) were administered intranasally (40 μL) as the first dose either 3 days before infection or 1 hour before infection or 1 day after infection (see dosing table below for more detail). First dose of 40 μL PBS (vehicle) will be given 1 hour before infection for both vehicle control (group 1) and virus only control (group 2).
Clinical signs for each animal were recorded once a day from Day −3. These included details on piloerection, respiration, activity, body posture, ocular/nasal discharge, body condition and ataxia. These variables were scored with the following severity bands:
Daily measurement of each animal's body weight was also recorded from Day −3.
Animals showing two or more of any of the limiting clinical signs (based on Home Office guidelines) equivalent to the protocol severity limit, were removed from the study and were culled by a schedule 1 method (cervical dislocation) at the establishment. Where an animal reached the limit of either or both of the first two signs with or without any other signs, it was removed from the study and culled by a schedule 1 method at the establishment.
Body weight loss greater than 20% of the highest measured individual body weight.
In this study, no animals were removed from the study on grounds of welfare issues.
4 days after infection, all animals were overdosed intraperitoneally with pentobarbitone, and blood samples were collected by venepuncture into Eppendorf-tubes (0.5 mL). Each sample was mixed gently and kept at room temperature for 30 min to allow clotting, before centrifugation (1500 rpm, 10 min at 4° C.) to prepare the serum. 2 aliquots (50 μL) of each sample were stored at −80° C. Immediately after collecting the blood, the trachea was isolated by a midline incision in the neck and separation of the muscle layers. A small incision was made into the trachea and a plastic cannula was inserted and secured in place with a suture. The airway was then lavaged by flushing out the lungs using 1 mL of phosphate buffered saline. This procedure was then repeated until the recovered volume was 1.6 mL. The isolated BALF was then be centrifuged at 1500 rpm for 10 mins at 4° C. and the supernatant was aliquoted (400 μL) at −80° C. for future cytokine analysis. The cell pellets were then re-suspended in 0.8 mL of 0.2% w/v NaCl to induce haemolysis of any erythrocytes. After isotonization with the same volume of 1.6% w/v NaCl, the BAL cells were analysed for total and differential numbers.
Total and differential cell counts of the BAL fluid samples were measured using a XT-2000iV analyser (Sysmex). Results are expressed as cells/mL (total and differential). Cell types differentially classified will be neutrophils, eosinophils or mononuclear cells (macrophages and lymphocytes).
Following BALF collection, the left and right lung lobes were removed and separated from each animal and homogenised in the volume of ×10 gram-lung weight (if 0.11 g, use 1.1 mL) of ice-cold Dulbecco's modified Eagles medium (DMEM containing 1% w/v BSA and 25% w/v sucrose) for 2×20 second bursts. The homogenate was transferred into a sterile tube and spun at 4° C. at 2000 rpm for 5 minutes. The clarified homogenate was then transferred to a chilled cryovial and snap frozen in liquid nitrogen and stored at −80° C.
HEp2 cells were grown in 24-well plates prior to infection in DMEM containing 10% v/v FBS until they attain 100% confluency. Lung homogenates were thawed out at room temperature and ten-fold serial dilutions were prepared in serum-free DMEM. The growth medium from HEp2 cells was aspirated and replaced with 300 μL of serially diluted lung homogenate (along with stock RSV only positive control) and left to infect at 37° C./5% CO2 for four hours. The infectious media was then aspirated and replaced with 500 μL Plaque Assay Overlay (1% w/v methylcellulose in MEM, 2% v/v FBS, 1% w/v pen/strep, 0.5 μg/ml amphotericin B), and left for 7 days at 37° C./5% CO2. Cells were fixed with ice-cold methanol for 10 minutes after which they were washed twice with sterile PBS. Anti-RSV F-protein antibody [2F7] was diluted to a 1:150 concentration in blocking buffer (5% w/v powdered milk (Marvel) in 0.05% v/v PBS-Tween 20) and 150 μL were added to cells for 2 hours at room temperature with shaking. Cells were washed 2× using PBS before 150 μl of secondary antibody (goat anti-mouse/HRP conjugate) diluted 1:400 in blocking buffer were added to cells for 1 hour at room temperature, with shaking. The secondary antibody solution was removed and cells were washed twice with PBS before the metal-enhanced development substrate DAB will be prepared in ultra-pure water (according to manufacturer's instructions). Each well received 150 μL of development substrate until plaques are visible. Plaques were counted by eye and confirmed using light microscopy, allowing the calculation of plaque-forming units per mL.
Cytokine levels (see below for details of cytokines evaluated) of BALF supernatant were measured in duplicate using magnetic multiplex assays as per the manufacturers' instructions. Levels were measured using a Magpix system (Luminex Corp.)
Data are reported as cytokine (pg/mL), mean±S.E.M. (standard error of the mean).
Data are reported as total and differential number of cells per mL of BALF, cytokine concentration (pg/mL) or plaque forming units (pfu) per treatment group, ±(standard error of the mean).
Inter-group deviations were statistically analyzed by a one-way analysis of variance (ANOVA). In the case of significant difference in the mean values among the different levels of treatment, comparisons versus the vehicle group will be carried out using the Dunnett's test. In case the equal variance test fails, a Kruskal-Wallis one-way analysis of variance on ranks followed by a Dunn's test will be proposed. p<0.05 were considered statistically significant.
Prophylactic treatment with Neumifil (HEX17) in RSV-A2 challenged mice resulted in a significant protective effect as evidenced by a reduction in lung tissue viral load. A strong effect was seen in mice which received a regimen of three doses of Neumifil on day 3, day 1 and 1 hour pre-challenge. Also, mice that received a single prophylactic dose 1 hour pre-challenge showed a statistically significant reduction in viral load. Mice that did not receive prophylactic treatment but were only treated post-challenge also showed a statistically significant reduction in lung tissue viral load albeit less than mice that received prophylactic treatment.
RSV-A2 infection in mice resulted in a strong immune response evidenced by changes in BAL fluid cell counts (total cell count, neutrophils and lymphocytes) and cytokines measured in BAL fluid. Groups of mice treated with Neumifil showed statistically significant improvements in a number of immune parameters; these results correlated well with the observed reductions in lung tissue viral loads.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1912365.2 | Aug 2019 | GB | national |
This application is a 35 U.S.C. § 371 national stage of PCT Application No. PCT/EP2020/074140, filed on Aug. 28, 2020, which claims priority from United Kingdom Patent Application No. 1912365.2, filed on Aug. 28, 2019, and to U.S. Provisional Patent Application No. 62/892,750, filed Aug. 28, 2019, the contents of each of which are incorporated herein by reference. The above-referenced PCT International Application was published in the English language as International Publication No. WO 2021/038080 A1 on Mar. 4, 2021.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/074140 | 8/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62892750 | Aug 2019 | US |
