Patent Grant
11389502
This application is a 35 U.S.C. § 371 national phase application of International Application Serial No. PCT/GB2017/052808, filed Sep. 20, 2017, which claims the benefit, of United Kingdom Patent Application No. 1616006.1, filed Sep. 20, 2016, the entire contents of each of which are incorporated by reference herein.
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 1476.6 Replacement Seq List ST25.txt, 25,202 bytes in size, generated on Mar. 9, 2022, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.
The present invention provides molecules for use in modulating cell growth and/or activity and for use in methods of treating or preventing cancer, its migration and/or spread.
It is well known that some lectins, which are glycoproteins of non-immune origin, exhibit an ability to induce apoptosis in malignant cells and thus demonstrate anti-cancer properties. This phenomenon occurs, in part, through the interaction of these lectins with specific glycan receptors on immune cells (Yau et al., 2015).
One such lectin is Viscumin from mistletoe, which is a toxin that binds to cellular receptors that are glycosylated with α2,6 sialyllactose (Müthing et al., 2002). Viscumin is a 57 kDa heterodimer, comprising of two subunits A and B. The A subunit exerts its toxic effect by disabling ribosomes, thereby interrupting protein production, whereas the B subunit exhibits glycan binding function. The lectin demonstrates picomolar cytotoxicity in vitro and in vivo, and has a recommended dose upper limit of 6 μg/kg in clinical trial subjects (half-life of 13 mins) (Zwierzina et al., 2011).
Another plant lectin that has demonstrated an ability to prevent cell migration and growth (and hence an anti-cancer property) is the Maackia amurensis seed lectin, or MASL (Ochoa-Alvarez et al., 2012; Astarita et al., 2012). This lectin is also cytotoxic exhibiting nanomolar potency (˜300 nM), and exerts its effects through the binding of podoplanin, an α2,3 sialylated mucin-type transmembrane glycoprotein, that is overexpressed in a variety of human cancers (Kato et al., 2005; Schacht et al., 2005; Shibahara et al., 2006).
The treatment of cell proliferation and differentiation disorders, including, for example cancer, demands the provision of additional molecules that are well tolerated in their hosts and have therapeutic potential.
The present disclosure is based on the finding that molecules which bind sialic acid also modulate aspects of cell growth and/or cell activity.
Throughout this specification, the terms “comprise”, “comprising” and/or “comprises” is/are used to denote that aspects and embodiments of this invention “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.
In a first aspect, there is provided a sialic acid binding molecule for use in a method of modulating cell growth and/or cell activity.
In a second aspect, there is provided a method of modulating cell growth and/or activity, said method comprising contacting a cell with a sialic acid binding molecule. The method may be an in vitro method.
The term “modulating” may embrace any increase or decrease in one or more aspects of cell growth and/or activity. In other words, a sialic acid binding molecule described herein may either inhibit certain aspects of cell growth and/or activity or may induce or stimulate other aspects of cell growth and/or activity.
The terms “growth” and “activity” as applied to cells may embrace processes and/or phenomena associated with one or more of cell proliferation, cell viability, cell migration, cell metabolism, cell differentiation and/or cell morphology/phenotype. The terms “growth” and/or “activity” may further include the response of a cell to certain exogenous and/or endogenous factors or stimuli including, for example, responses to certain compounds of the immune system, cytokines, chemokines and one or more environmental factors (light, temperature, pressure, mechanical stress and the like). Thus, the sialic acid binding molecules disclosed herein may be used to modulate (inhibit, decrease or increase) levels of cell responsiveness.
Given that sialic acid binding molecules have been shown to modulate cell growth and activity (as described above), it will be appreciated that there are also a number of related medical and veterinary applications and uses for these sialic acid binding molecules. For example, a sialic acid binding molecule may be applied to the treatment and/or prevention of diseases in which aberrant cell growth, and/or aberrant cell activity is a factor.
Thus, there is provided a sialic acid binding molecule for use in treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or activity.
Further, there is provided use of a sialic acid binding molecule for the manufacture of a medicament for the treatment and/or prevention of a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or activity.
There is also provided a method of treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or activity, said method comprising the step of administering a therapeutically effective amount of a sialic acid binding molecule to a subject in need thereof.
Diseases which are caused, contributed to or characterised by aberrant cell growth and/or activity may include, for example cell proliferation and/or differentiation disorders including, those referred to or classified as benign or malignant conditions. For example, the term “cell proliferation and/or differentiation disorders” may include those diseases and/or conditions collectively referred to as “cancer”. The term “cancer” may include, but is not limited to, those cancers referred to as forms of breast cancer, bone cancer, brain cancer (gliomas), pancreatic cancer, lung cancer, prostate cancer, skin cancer, ovarian cancer, cervical cancer, head and neck cancers and bowel/colon cancer. The term “cancer” may also include those diseases and/or conditions collectively referred to as “leukaemias” (both chronic and acute) and any cancer affecting a mucosal/mucosal associated surface or tissue.
As such, a sialic acid binding molecule described herein may find application in the treatment and/or prevention of cancer.
Thus, there is provided a sialic acid binding molecule for use in treating and/or preventing cancer.
Further, there is provided use of a sialic acid binding molecule for the manufacture of a medicament for the treatment and/or prevention of cancer.
There is also provided a method of treating and/or preventing cancer, said method comprising the step of administering a therapeutically effective amount of a sialic acid binding molecule to a subject in need thereof.
A subject in need thereof or indeed a subject to be administered a sialic acid binding molecule disclosed herein or a medicament comprising the same, may be any subject suffering (or suspected as suffering) from (i) a cell proliferation and/or differentiation disorder, (ii) cancer, (iii) any other disease and/or condition described herein; or (iv) a disease or condition caused, contributed to or characterised by aberrant cell growth and/or activity. Additionally, or alternatively, any subject may be a subject predisposed or susceptible to (i) a cell proliferation and/or differentiation disorder, (ii) cancer, (iii) any other disease and/or condition described herein; or (iv) a disease or condition caused, contributed to or characterised by aberrant cell growth and/or activity.
It should be understood that the treatment of a cell proliferation and/or differentiation disorder, a cancer or a disease or condition caused or contributed to (or characterised by) aberrant cell growth and/or activity, may involve the use of one or more sialic acid binding molecule(s) of this disclosure to treat, ameliorate or reduce, one or more symptoms of those diseases. By way of example, the symptoms of a disease such as cancer may include, for example, the presence of tumours and/or cell masses. As such, the sialic acid binding molecules described herein may be used to modulate (for example stop, retard, inhibit or reduce) tumour formation and/or the metastasis thereof. The sialic acid binding molecules may also be used to reduce the overall size of a tumour. Certain tumours, including those that are large and/or aggressive, are often easier to surgically remove if they have first been reduced in size. Typically, chemo- and/or radiotherapy based treatments might be used to reduce the size of a tumour but treatments such as this may be replaced by and/or supplemented with sialic acid binding molecule based treatments. As stated, molecules which exhibit sialic acid molecule binding activity exhibit an ability to modulate cell growth and/or activity and therefore, without wishing to be bound by theory, the mechanism underpinning the ability of a sialic acid binding molecule to affect the size of a tumour may be rooted in the cell proliferation, differentiation and/or metabolism modulating effects of the molecule.
In view of the above, the successful treatment of a tumour may therefore be characterised by a reduction in tumour size, a reduction in an observed or detectable/detected level of tumour metastasis, angiogenesis within tumorigenic tissue and/or tissue invasion.
Thus, sialic acid binding molecules may be for use in methods of modulating (for example inhibiting, restricting or reducing) tumour growth, development and/or metastasis in subjects in need thereof. The sialic acid binding molecules described herein may be formulated as compositions for use in modulating tumour growth, development and/or metastasis or used in the manufacture of medicaments for achieving the same. This disclosure also provides a sialic acid binding molecule for use in treating a tumour. Further, described is the use of a sialic acid binding molecule for the manufacture of a medicament for treating a tumour. Also, the disclosure provides a method of treating a tumour, said method comprising administering a sialic acid binding molecule to a subject (or tumorigenic tissue) in need thereof.
As defined earlier, the term “a subject in need thereof” may embrace any subject suspected as having a tumour or diagnosed with a tumour and/or subjects that are identified as being predisposed and/or susceptible to tumours.
The present disclosure therefore provides various applications for sialic acid binding molecules which have been identified as modulators of cell growth and/or activity. Any given sialic acid binding molecule may be identified as a modulator of cell growth and/or activity via the various experiments and assays described in the examples section of this patent application. For example, the cell wound scratch assay is one example of an assay that may be adapted to determine whether any given “test” molecule exhibits the necessary ability to modulate cell growth and/or activity.
Thus, the invention relates to those sialic acid binding molecules which via a cell activity modulation assay (for example a cell wound scratch assay) exhibit an ability to modulate cell growth and/or activity. To this end, the disclosure further provides a method of identifying sialic acid binding molecules for use in methods of modulating cell growth and/or activity or for the various medical and/or veterinary applications described herein, said method comprising subjecting a test compound to an assay capable of reporting an effect of the test compound on cell growth and/or activity, wherein the test compound is a sialic acid binding compound and any if the assay reports that the compound has an effect on any aspect of cell growth and/or activity, the compound may be useful in the treatment and/or prevention of diseases and/or conditions of the type described herein. The assay which is capable of reporting an effect of the test compound on cell growth and/or activity may be a cell wound scratch assay as described herein.
The present disclosure relates to molecules and compounds which exhibit an ability to bind to sialic acid. These 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 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 GalNAc 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—including all forms of sialic acid described above and any form of sialic acid present on the surface of a cell, 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 any fragment of a whole sialic acid binding molecule which retains an ability to bind to or otherwise couple or associate with a sialic acid moiety.
Sialic acid binding molecules for use 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 for use 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. Carbohydrate binding modules 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.
Exemplary carbohydrate binding modules for use 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 or 2 or a sialic acid binding fragment thereof.
A useful sialic acid binding molecule may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the nanH gene (encoding sialidase) of V. cholerae (as provided by SEQ ID NO: 1) or an equivalent or homologous gene present in another organism (for example the equivalent/homologous nanA sialidase gene of S. pneumoniae: see SEQ ID NO: 3).
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 further suitable sialic acid binding molecule may comprise a protein or peptide having the sequence of SEQ ID NO: 3 or 4 or a sialic acid binding fragment thereof. For example, a useful sialic acid binding molecule may comprise a proteinaceous moiety encoded by the sialic acid binding domain of the Streptococcus pneumoniae nanA gene (encoding sialidase). For example, 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 CBMs—for example, a single VcCBM or a single SpCBM. 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 VcCBMs or two or more SpCBMs. A multivalent CBM may comprise a mixture of different CBMs, for example one or more VcCBMs with one or more SpCBMs.
For example, multivalent CBM molecules, (for example a Vc4CBM molecule) may be prepared as constructs comprising multiple CBMs linked by amino acid/peptide linkers. Each CBM (for example VcCBM) 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 to produce a multivalent CBM:
Thus, the various aspects and embodiments of this invention (uses, sialic acid binding molecules for use, methods and medicaments) may exploit sialic acid binding molecules which comprise, consist of or consist essentially of sialic acid binding molecules selected from the group consisting of:
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 CBMs) 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 PAO579 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 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.
A sialic acid binding molecule for use may comprise one or more of the CBM based molecules presented in
Other sialic acid binding domains for use 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 for use 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
Thus, this disclosure provides:
(i) a CBM for use in a method of modulating cell growth and/or cell activity; said method comprising contacting a cell with a CBM;
(ii) a method of modulating cell growth and/or activity, said method comprising contacting a cell with a CBM. The method may be an in vitro method;
(iii) a CBM for use in treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or proliferation;
(iv) use of a CBM for the manufacture of a medicament for the treatment and/or prevention of diseases and/or conditions caused, contributed to and/or characterised by aberrant cell growth and/or proliferation;
(v) a method of treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or proliferation, said method comprising the step of administering a therapeutically effective amount of a sialic acid binding molecule to a subject in need thereof;
(vi) a CBM for use in treating and/or preventing cancer;
(vii) use of a CBM for the manufacture of a medicament for the treatment and/or prevention of cancer; and
(viii) a method of treating and/or preventing cancer, said method comprising the step of administering a therapeutically effective amount of a sialic acid binding molecule to a subject in need thereof.
In any one of embodiments (i) to (viii) above, the CBM may be a multivalent CBM comprising two or more family 40 CBMs. For example, in any one of embodiments (i) to (viii) above, the CBM may comprise (consist essentially of, or consist of) two or more VcCBMs and/or SpCBMs optionally fused, bound or conjugated to an oligomerisation domain (such as a PaTD or oligomerisation fragment thereof). The sialic acid binding molecule may comprise, consist or consist essentially of two fused (or bound) VcCBMs or two fused (or bound) SpCBMs which are in turn fused to an oligomerisation domain (see, for example, molecule Vc2CBMTD and Sp2CBMTD shown in
In view of the above, this disclosure provides:
(i) the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) for use in a method of modulating cell growth and/or cell activity; said method comprising contacting a cell with the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM);
(ii) A method of modulating cell growth and/or activity, said method comprising contacting a cell with the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM). The method may be an in vitro method;
(iii) The sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) for use in treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or proliferation;
(iv) use of the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) for the manufacture of a medicament for the treatment and/or prevention of diseases and/or conditions caused, contributed to and/or characterised by aberrant cell growth and/or proliferation;
(v) a method of treating and/or preventing a disease and/or condition caused, contributed to and/or characterised by aberrant cell growth and/or proliferation, said method comprising the step of administering a therapeutically effective amount of the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) to a subject in need thereof;
(vi) the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) for use in treating and/or preventing cancer;
(vii) use of the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) for the manufacture of a medicament for the treatment and/or prevention of cancer; and
(viii) a method of treating and/or preventing cancer, said method comprising the step of administering a therapeutically effective amount of the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) to a subject in need thereof.
Additionally, the disclosure provides:
(i) A CBM (as described herein) and/or the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) for use in (a method of) modulating tumour growth;
(ii) Use of a CBM (as described herein) and/or the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM) in the manufacture of a medicament for use in modulating tumour growth; and
(iii) A method of modulating tumour growth, said method comprising administering a therapeutically effective or tumour growth modulating amount of a CBM (as described herein) and/or the sialic acid binding domain of Vibrio cholerae NanH sialidase (VcCBM) and/or the sialic acid binding domain of Streptococcus pneumoniae NanA sialidase (SpCBM), to a subject in need thereof.
Molecules which bind sialic acid and in particular sialic acid which is part of a cell bound receptor, may find further application as moieties which may be conjugated, bound or joined to or associated with, other entities for the purpose of targeting or delivering that entity to some tissue or cell. Molecules of this type may be otherwise known as “therapeutic warheads” or “conjugates”. Without wishing to be bound by theory, the presence of sialic acid in certain cell receptors and membrane bound molecules, may allow the sialic acid binding molecule to be exploited as a means to deliver conjugated heterologous molecules (that is a molecule distinct from and different to the sialic acid binding molecule) to said cells or tissues comprising said cells. Thus, conjugated sialic acid binding molecules may be useful in the treatment of cancer, where sialic acid binding molecules (with affinity for sialic acids expressed on cancerous cells and/or tumours) may be used to direct therapeutic and/or cytotoxic moieties thereto.
By way of example, a sialic acid binding molecule as described herein (including any of the CBM based molecules) may be conjugated to moieties which are, for example, therapeutic and/or cytotoxic. Thus, the disclosure relates to sialic acid binding molecule conjugates.
Sialic acid binding molecule conjugates may comprise a sialic acid binding molecule of this disclosure conjugated (joined, bound or otherwise associated with) to a heterologous moiety. The heterologous moiety may comprise a therapeutic and/or cytotoxic moiety which may be conjugated to some part of the sialic acid binding molecule. For example, the heterologous moiety may be conjugated to one or both ends of the sialic acid binding molecule. The heterologous moiety may be additionally or alternately conjugated (or even fused) to an internal portion of the sialic acid binding molecule. It will be appreciated that however the heterologous moiety is to be conjugated to the sialic acid binding molecule, the moiety (nor its conjugation) should not (substantially) interfere with or ablate or reduce the sialic acid binding property of the sialic acid binding molecule.
As stated, the heterologous moiety may be a drug useful in the treatment of a disease which affects a cell or tissue comprising sialic acid. For example, the drug may be a chemotherapeutic drug for use in the treatment of cancer and the like. The heterologous moiety may be a cytotoxic moiety capable of killing or inducing apoptosis in, a cell. The heterologous moiety may comprise a molecule which is able to recruit specific cells to or into a particular tissue. For example, the heterologous moiety may be, for example, a T cell receptor (TCR) which may be used as a means to recruit T cells to, for example a tumour or cancerous tissue.
The present disclosure may provide compositions for use in the various uses, medicaments and methods described herein. As such, any of the sialic acid binding molecule(s) described herein may be formulated for use. For convenience, and with reference to the section below describing compositions, formulations and the like it should be noted that both sialic acid binding molecules as described herein and any conjugates comprising the same (for example sialic acid binding molecule:drug conjugates/fusions) shall be included under the general term “sialic acid binding molecule”.
A sialic acid binding molecule (or molecules) may be formulated for use and as a therapeutic or pharmaceutical composition. 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.
The various sialic acid binding molecules described herein may be formulated for enteral (including oral), parenteral and/or topical administration and one of skill will appreciate that the precise formulation may vary depending on the route of administration.
Pharmaceutical compositions according to the present invention 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.
A therapeutic or pharmaceutical composition of this disclosure (that is a composition comprising a sialic acid binding molecule and for use in any of the medicaments or methods described herein—including the methods of or medicaments for, modulating cell growth and/or activity and/or treating cancer) may be formulated together with one or more pharmaceutically acceptable excipients, carriers, adjuvants and buffers. The compositions can be administered, e.g. orally (including mucosally), parentally, enterally, intramuscularly, subcutaneously, intravenously or via any other routes useful to achieve the desired effect (in this case effects which include, modulation of cell growth/activity, treatment or prevention of diseases/conditions associated with the same and/or cancer and/or modulation of tumour growth). As stated, depending on the chosen route of administration, the exact composition of the formulation may vary.
A therapeutic or pharmaceutical formulation comprising a sialic acid binding molecule and for administration to a subject may be coated, encapsulated or enveloped in a material which protects the sialic acid binding molecule from the action of enzymes, acids and other natural compounds/conditions (including, for example, compounds (including antibodies), cells and processes of the immune system) which may inactivate or denature the compound and/or its sialic acid binding properties.
Among the various standard and conventional excipients that may be available for use in compositions comprising sialic acid binding molecules, are those pharmaceutically acceptable organic or inorganic carrier substances which are suitable for parenteral, enteral, oral (including mucosal) and other routes of administration that do not deleteriously react with the sialic acid binding molecule(s).
Where the sialic acid binding molecules are to be formulated for parental administration, the compositions may be sterile.
The composition may comprise an oil-based or aqueous solution, a suspension and/or an emulsion.
In other embodiments, the composition may take the form of an implant, such as for example a (dissolvable or biodegradable) film, pessary or implant (including suppositories).
The pharmaceutical preparations comprising sialic acid binding molecules may be mixed with stabilizers, wetting agents, emulsifiers, salts (for use in influencing osmotic pressure), buffers and/or other substances that do not react deleteriously with the active compounds.
One or more of the sialic acid binding molecules described herein may be formulated for and administered, orally. As stated, oral administration would include mucosal administration which would itself would include administration intranasally and/or by inhalation.
Compositions for use may include solid dosage forms which are suitable for oral administration. These may include, for example capsules, tablets, pills, powders, and granules. In any given solid dosage form, the sialic acid binding molecule (or any conjugate comprising the same) may be admixed with at least one inert pharmaceutically-acceptable excipient. Examples of suitable excipients will be known to one of skill in this field but may include, for example fillers or extenders, humectants, wetting agents, binders, disintegrating agents, solution retarders, absorption accelerators, adsorbents, lubricants or mixtures thereof. A tablet, pill or capsule may further comprise a buffering agent. Solid dosage forms such as tablets, dragees, capsules, pills and/or granules also can be prepared with coatings and shells, such as coatings which protect against the gastrointestinal environment and/or stomach acid.
A solid dosage form may contain opacifying agents, and can also be formulated so as to ensure the delayed release of the active agent (in this case the sialic acid binding molecule or a conjugate comprising the same) in or to a specific part of the intestinal tract.
Solid compositions for oral administration can be formulated in a unit dosage form, each dosage containing an appropriate dose of the sialic acid binding molecule (or conjugate comprising the same). The exact amount of sialic acid binding molecule (or conjugate comprising the same) contained within any given solid dosage form will vary depending on the intended use. a solid composition may contain a “unit dose”—a unit dose containing a quantity of sialic acid binding molecule (or conjugate containing the same) calculated to produce the desired effect (for example modulation of cell growth and/or activity) over the course of a treatment period.
Liquid dosage forms for oral administration may (as stated) include emulsions, solutions, suspensions, syrups, and elixirs. In addition to the compound or composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers.
The sialic acid binding molecules may be used in any suitable amount. As stated, the sialic acid binding molecules may be formulated for oral, mucosal or parenteral administration and as such, the precise formulation may depend on the intended route of administration. The amount of sialic acid binding molecule present in any given dose may be in the region of 0.1 μg-1000 μg. For example, amounts of about 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 1 μg, 10 μg, 20 μg, 25 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg or 900 μg. The selected amount of sialic acid binding molecule may be formulated in a specific volume of a pharmaceutically acceptable excipient, diluent and/or buffer. The volume of excipient, diluent or buffer may be about 10 μl to 5 ml. For example, the required amount of sialic acid binding molecule (CBM) may be combined (or formulated) with about 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, 250 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 ml, 2 ml, 3 ml or 4 ml. For example, an amount of 100 μg sialic acid binding molecule may be combined with about 250 μl of excipient to yield a final concentration of sialic acid concentration of 400 μg/ml. Doses at concentrations of about 0.1 μg/ml-1 mg/ml may be used including, for example, doses at 5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml or 900 μg/ml.
In use, a dose of a sialic acid binding molecule, administered as part of the treatment and/or prevention of a cell proliferation and/or differentiation disorder (for example cancer), may be administered multiple times over a number of days or weeks. For example, after an initial (or first) administration, a dose of a sialic acid binding molecule may be administered again at about (+/−1 or 2 days) 3, 4, 5, 6, 7, 17, 21, 28 and/or 35 days later. On any given day, a specific dose of a sialic acid binding molecule may be administered 1, 2, 3 or more times. Each time, the sialic acid binding molecule may be administered (by whatever route is considered best to affect a suitable treatment or to induce prophylaxis against the development of a cell proliferation and/or differentiation disorder.
The present invention will now be described in detail by reference to the following Figures which show:
In an in vitro mCBM40 dosing experiment using confluent A549 human lung carcinoma cells and high concentrations of Sp2CBMTD (400-800 μg), little or no cell growth or activity was observed after 3 days. This was noted as the colour of the culture medium (DMEM/5% FCS), which would change colour as it is utilised, remained the same (see
This observation led to the hypothesis that our engineered sialic acid-recognising CBMs might affect cell (including cancer cell) proliferation and migration by targeting sialylated host cell receptors.
To further support this hypothesis a wound scratch assay to determine cell migration and proliferation and a cell viability (MTT) assay were performed on A549 human lung carcinoma cells. These cells were treated with CBMs for 24 h. The results were compared to control cells that were given PBS only.
As seen in
Metabolic activity decreased in cells that were given Vc2CBMTD in serum-free medium (absence of FCS), compared to cells that were given PBS only. Cell morphology of Vc2CBMTD-treated cells was also observed to have changed, with cells appearing more rounded (a sign of cell arrest) when compared to the control. Interestingly, cell metabolic activity and phenotype appeared to be reversed with the addition of 2 mM sialyllactose. Similar concentrations of sialic acid can also be found in FCS, and this may be the reason why Vc2CBMTD-treated cells remained viable after 3 days compared to Sp2-treated cells, which appear to be unaffected by the presence of FCS, at least at the high dose of CBM used in the assay (
As for Sp2CBMTD-treated cells, there was also a slight (but not significant) decrease in metabolic activity. However, the lack of significance is likely due to the low concentration administered to the cells.
There was a slight recovery of cell growth in the presence of sialyllactose compared to Vc2-treated cells, but due to the nature of Sp2CBMTD, which is also known to stimulate pro-inflammatory mediators, it is likely that the phenomenon observed in
Wound Healing and Proliferation Assay of Cancer Cell Lines
Method:
All CBM40 proteins were prepared as described in Connaris et al (2014)1. The following cancer cell lines, A549 (human lung), HeLa (cervical), MDA.MB.231 and MCF-7 (breast), and SW620 and SW480 (colon) were purchased from ATCC. All cells with the exception of HeLa cells were maintained in 10% FBS, DMEM (high glucose with 1% Penicillin/Streptomycin) and incubated at 37° C. and 5% C02. HeLa cells expressing GFP restricted to the nucleus (using IncuCyte NucLight Green Lentivirus Reagent Cat No. 4626 Essen Biosciences) were maintained in complete medium containing 0.5 μg/ml Puromycin. For wound healing assays, 24 well plates (Nunc) were seeded with cells (2.5×105 cells/well) and maintained in 500 μL of their respective maintenance medium and incubated at 37° C. and 5% CO2 for 24 hrs. Wells were washed with 500 μL of serum-free DMEM, prior to the wound/scratch creation. A wound/scratch was created as a straight line down the middle of the well (running from the top to bottom of the well) in each well using a sterile 200 μL pipette tip (Axygen). Wells were then washed with 500 μL of serum-free DMEM prior to the addition of 250 μL of appropriately diluted CBMs, also prepared in serum-free DMEM. Serum-free medium was also included to wells as a control. Plates were incubated at 37° C. and 5% CO2 for 10-30 mins before a further addition of 250 μL of 4% FBS, DMEM to each well (2% FBS final concentration) so that the final CBM concentration ranged from 20-200 μg/ml. The IncuCyte ZOOM (Essen BioScience) apparatus was used to collect time-lapse images of each well. This was set to collect three images of the wound/scratch in each well every 1 hr for 72 h. For data collection, the IncuCyte ZOOM 2016 software was used to measure and collate wound width measurements for each image (3× wound measurements were performed manually). For the cell proliferation assay, GFP-labelled HeLa cells were grown in 24 well plates over 72 h in the presence or absence of mCBM40s. Cell proliferation is monitored by analysing the occupied area (percentage of confluence) of cell images over time using the IncuCyte ZOOM apparatus. Cell proliferation is directly proportional to increase in confluence.
Results:
The inhibition of wound healing was due to the lack of cell proliferation over time as shown in
To determine whether the anti-proliferative effect is a result of the multimeric nature of engineered CBMs, A549 and HeLa cell lines were treated with the monomeric versions of Vc- and Sp-based CBM40s (VcCBM, SpCBM, 100 μg each) and left to incubate for up to 72 h. The data was then compared with that of their multimeric counterparts.
Furthermore, the non-CBM40 component of mCBM40s, that is, the trimerisation domain (TD), was also tested in a scratch assay to determine if this domain may contribute to the anti-proliferative effect seen in the scratch assays.
To establish if anti-proliferative activity of CBM proteins can be reversed after a wound scratch assay, a wash-off experiment was attempted. After 72 h incubation, treated wells were washed twice with 0.5 mL of 2% FBS, DMEM (high glucose, 1% Penicillin/Streptomycin). The same medium (0.5 mL) was then added to each well and incubated for a further 72 h. The EVOS FL (ThermoFisher Scientific) apparatus was used to manually collect three images of the wound/scratch in each well at 0 hrs and 72 hrs. Image J software was used to manually measure and collate wound width measurements for each image (Image J measurement tool was calibrated against a scale bar for the EVOS FL). After removal of mCBM40s, cells were clearly seen to migrate and/or proliferate into the wound (
Migration Assay.
Method:
A Boyden chamber assay was selected to determine if cellular migration is interrupted by multimeric CBM40s. The Boyden chamber assay (8 μm, fluorometric format) was prepared as described in manufacturer's instructions (Cell Biolabs CytoSelect 96-well cell migration assay). Final CBM concentration ranged from 20-200 μg/ml.
Results:
Cell Apoptosis, Viability and Cytotoxicity Assay.
Method:
To determine if multimeric CBMs cause cell apoptosis, the Promega ApoTox-Glo™ Triplex Assay was used. This assay was performed using A549 cells. 10,000 cells per well were seeded 24 hrs prior to experiment. CBMs and controls were added to wells to a final volume of 100 μL with final CBM concentrations of 0.1-1 mg/ml. Cells were cultured in conditions for test exposures of 4 hrs, 24 hrs, 48 hrs and 72 hrs (
Results:
After 48 hrs, Vc2CBMTD (at 400 μg) showed elevated luminescence signal corresponding to activation of caspase 3 (a hallmark of apoptosis). All the other conditions at 4 hrs, 24 hrs and 48 hrs showed similar luminescence levels to that of the untreated cells (
Discussion
The results identify that CBMs (Vc4CBM, Vc2CBMTD and Sp2CBMTD), but not the non-CBM component (TD), inhibit cellular behaviours required for wound healing (proliferation and migration) and are effective against multiple cancer types as indicated by experiments using breast, colon, cervical and lung cells. Agglutination of the A549 cell line was not observed. The inhibition of both migrating and proliferating cells in the wound-healing assay can be reversed via a wash-off. The results obtained from the Promega ApoTox-Glo™ Triplex Assay suggest that CBMs cause cell cycle arrest/or dormancy in the A549 cell line.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1616006 | Sep 2016 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2017/052808 | 9/20/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/055373 | 3/29/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20020025320 | Boyaka et al. | Feb 2002 | A1 |
| 20040072256 | Mandelboim et al. | Apr 2004 | A1 |
| 20050084903 | Kim et al. | Apr 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| 2799086 | Nov 2014 | EP |
| 0010388 | Mar 2000 | WO |
| 2002094869 | Nov 2002 | WO |
| 2007075921 | Jul 2007 | WO |
| 2008053486 | May 2008 | WO |
| WO-2009049234 | Apr 2009 | WO |
| 2010005737 | Jan 2010 | WO |
| 2010029312 | Mar 2010 | WO |
| 2010052492 | May 2010 | WO |
| 2010102112 | Sep 2010 | WO |
| 2014052621 | Apr 2014 | WO |
| 2015110831 | Jul 2015 | WO |
| WO-2018006034 | Jan 2018 | WO |
| 2018055365 | Mar 2018 | WO |
| 2018055370 | Mar 2018 | WO |
| Entry |
|---|
| UniprotKB-P0C6E9 Mar. 18, 2008. |
| Chatterjee et al. Cancer Immunol Immunother. 1994 38:75-82. |
| Corcos et al. Cancer Medicine 2013; 2(4):421-426. |
| Ferreira, Daniela & Adega, Filomena & Chaves, Raquel. (2013). The Importance of Cancer Cell Lines as in vitro Models in Cancer Methylome Analysis and Anticancer Drugs Testing. 10.5772/1745. |
| Chakraborty et al. ecancer 2012, 6:ed16 DOI:10.3332/ecancer.2012.ed16. |
| Boraston et al Biochemistry 2007, 46, 11352-11360. |
| Baradaran et al. “Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent”, Journal of Cancer, 7,(4):462-466 (2016). |
| Chen at al, “Preserving Sialic Acid-dependent Pattern Recognition by CD24-Siglec G Interaction for Therapy of Polybacterial Sepsis” Nature Biotechnology, 29(5):428-435 (2011). |
| Connaris et al. “Enhancing the Receptor Affinity of the Sialic Acid-binding Domain of Vibrio cholerae Sialidase through Multivalency” Journal of Biological Chemistry, 284(11):7339-7351 (2009). |
| Connaris et al. “Prevention of influenza, by targeting host receptors using engineered proteins” Proceedings of the National Academy of Sciences, 111(17):6401-6406 (2014). |
| Gasiorowski et al. “The impact of neuraminidase on apoptosis in cultures of blood lymphocytes isolated from rats bearing morris hepatoma” Cellular & molecular biology letters, pp. 389-399, URL: http://www.cmbl.org.pl/pdf/Vol9_p389.pdf (2004). |
| Govorkova et al. “Sialic Acid-Binding Protein Sp2CBMTD Protects Mice against Lethal Challenge with Emerging Influenza A (H7N9) Virus” Antimicrobial Agents and Chemothe, American Society for Microbiology, 59(3):1495-1504 (2015). |
| Grata-Borkowska et al. “Effects of neuraminidase on apoptosis of blood lymphocytes in rats with implanted Morris tumor” Journal of physiology and pharmacology: an official journal of the Polish Physiological Society, p. 253, URL: http://www.jpp.krakow.pl/journal/archive/11_07_s5/pdf/253_11_07_s5_article.pdf (2007). |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/GB2017/052800, dated Mar. 26, 2019, 6 pages. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/GB2017/052805, dated Mar. 26, 2019, 8 pages. |
| International Preliminary Report on Patentability corresponding to International Patent Application No. PCT/GB2017/052808, dated Mar. 26, 2019, 9 pages. |
| Manco et al. “Pneumococcal Neuraminidases A and B Both Have Essential Roles during Infection of the Respiratory Tract and Sepsis” Infection and Immunity, 74(7):4014-4020 (2006). |
| Simmons et al, “Immunospecific Regression of Methylcholanthrene Fibrosarcoma With the Use of Neuraminidase, II, Intratumor Injections of Neuraminidase” SUR, 71(4):556-564 (1972). |
| Simmons et al. “Regression of Established Methylcholanthrene Tumors By Intratumor Injections of Vibrio Cholerae Neuraminidase” Journal of Surgical Onco, 4(4):298-305 (1972) Abstract. |
| Written Opinion and International Search Report corresponding to International Patent Application No. PCT/GB2017/052800, dated Mar. 29, 2018, 10 pages. |
| Written Opinion and International Search Report corresponding to International Patent Application No. PCT/GB2017/052805, dated Mar. 29, 2018, 11 pages. |
| Written Opinion and International Search Report corresponding to International Patent Application No. PCT/GB2017/052808, dated Mar. 29, 2018, 16 pages. |
| Yang et al. “Structural characterization of the carbohydrate-binding module of NanA sialdase, a pneumococcal virulence factor” BMC Structural Biology, 15(1):15(2015). |
| Alias “Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection” PhD Thesis at University of St Andrews, 252 pages (2013). |
| Knop “Stimulatory effect of Vibrio cholera neuraminidase on the antibody response to various antigens” Immunology, 34:181-187 (1978). |
| Research Councils UK, Gateway to Research “Exploiting a sialic acid binding domain” University of St Andrews, 4 pages, accessed Jun. 1, 2017 from http://gtr.rcuk.ac.uk/projects?ref=BB%FE001912%2F1. |
| Rios et al. “Experimental Cancer Immunotherapy: modification of tumor cells to increase immunogenicity” Annals New York Academy of Sciences, 276:45-60 (1976). |
| Simmons et al. “Immunospecific Regression of Methylcholanthrene Fibrosarcoma With the Use of Neuraminidase: III. Synergistic Effect of BCG and Neuraminidase Treated Tumor Cells” Ann. Surg., 176 (2):188-194 (1972). |
| Crost et al. “The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular transsialidases” Gut Microbes, 7(4):302-312 2016. |
| Lipniĉanová et al. “Diversity of sialidases found in the human body—A review” Intl. J. Biol. Macromolecules, 148:857-868 2020. |
| Büll, Christian , et al., “Sialic Acids Sweeten a Tumor's Life”, Cancer Research 74(12), 2014, 3199-3204. |
| Dobie, Christopher , et al., “Insights into the roll of sialylation in cancer progression and metastasis”, British Cancer Journal 124, 2021, 76-90. |
| Pearce, Oliver M T, et al., “Sialic Acids in Cancer Biology and Immunity”, Glycobiology 26(2), 2016, 111-128. |
| Number | Date | Country | |
|---|---|---|---|
| 20190247460 A1 | Aug 2019 | US |
