GB2562691A

GB2562691A - Methods for the treatment and prevention of Ebola

Info

Publication number: GB2562691A
Application number: GB1617715.6A
Authority: GB
Inventors: Aljamal-Naylor Rehab; Soloveva Veronica; Bavari Sina
Original assignee: Avipero Ltd; Rehab Aljamal Naylor
Current assignee: Avipero Ltd; Rehab Aljamal Naylor
Priority date: 2016-10-19
Filing date: 2016-10-19
Publication date: 2018-11-28
Also published as: GB201617715D0

Abstract

A method for inhibiting and/or treating ebolavirus (EBOV) infection comprising administering a therapeutically effective amount of Ezetimibe or its derivatives alone, or in combination with, a compound which mediates allosteric modulation of beta 1 integrin. The compound which mediates allosteric modulation of beta 1 integrin may be an antibody that binds to an epitope present on the extracellular domain of beta 1 integrin such as the hybrid domain. The antibody may be HUTS21 or B44. Also claimed is a compound that binds to at least one epitope on the extracellular domain of beta integrin 1 and an assay method for identifying compounds for use in the inhibition and/or treatment of EBOV comprising screening candidate compounds for the ability to allosterically modify beta 1 integrin and compounds which affect sphingomyelinase function.

Description

(54) Title of the Invention: Methods for the treatment and prevention of Ebola Abstract Title: Method for the treatment and prevention of Ebola (57) A method for inhibiting and/or treating ebolavirus (EBOV) infection comprising administering a therapeutically effective amount of Ezetimibe or its derivatives alone, or in combination with, a compound which mediates allosteric modulation of beta 1 integrin. The compound which mediates allosteric modulation of beta 1 integrin may be an antibody that binds to an epitope present on the extracellular domain of beta 1 integrin such as the hybrid domain. The antibody may be HUTS21 or B44. Also claimed is a compound that binds to at least one epitope on the extracellular domain of beta integrin 1 and an assay method for identifying compounds for use in the inhibition and/ or treatment of EBOV comprising screening candidate compounds for the ability to allosterically modify beta 1 integrin and compounds which affect sphingomyelinase function.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

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Figure 4a. Average % Inhibition:

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Methods for the treatment and prevention of Ebola

Field of the Invention

The present invention provides a method for the treatment and/or prevention of Ebola viral infections. In particular, the invention provides a method for inhibiting and/or treating Ebola viral infections comprising a step of administering a therapeutically effective amount of an azetidinone-based cholesterol absorption inhibitor particularly ezetimibe, one of its analogues or its benzylic glucuronide either alone or in combination with a therapeutically effective amount mediates allosteric modulation of beta 1 integrin and to a subject in need thereof. Typically the allosteric modulation of beta 1 integrin results in beta 1 integrin assuming an intermediate affinity state partially activated conformation. The method of the present invention may be used in the treatment and/or prevention of Ebola viral infections.

Background to the Invention

Integrins constitute a family of widespread heterodimeric cell surface receptors composed of alpha (a) and beta (β) subunits. Integrins are grouped into four distinct subfamilies based upon beta subunit utilisation. Members of the beta 1 subfamily contain a common beta 1 chain (CD29). Human CD29 is expressed on diverse cell types which function as the major receptors for the extracellular matrix (ECMj and as cell-cell adhesion molecules. The ECM interacts with integrins and modifies their function. In turn, integrins are key regulators of the ECM. Their main functions are cell-ECM and cell-cell adhesion. One hallmark feature is the ability of integrins to process signals bi-directionally across the cell membrane. Additionally, integrins are mechanotransducers, converting mechanical signals to biochemical changes. These properties mean integrins play a key role in cell growth, cell differentiation, cell migration and cell survival.

CD29 can form heterodimer pairs with at least nine different alpha subunits. Heterodimers of beta 1 integrin bind collagens (al, a2), laminins (al, a2, a3, a7, a9) and fibronectin (a3, a4, a5, a8, av). Integrins in general, including beta 1 integrin, exhibit global structural rearrangement and exposure of ligand binding sites upon activation. The overall strength of cellular adhesiveness, or avidity, is governed by affinity and valency (which is, in turn, governed by the density of the receptor and its ligand on the cell surface, as well as spatial and geometric arrangement and movement). Recent evidence has demonstrated that both affinity and avidity of integrins are strongly related to the size of the focal adhesion clusters.

The alpha and beta subunit of all integrins fold forming an extracellular headpiece which is connected to the membrane by a structure which can be likened to two supporting legs”, followed by a short transmembrane domain and a cytoplasmic tail. The headpiece of the integrin heterodimer is composed of a beta propeller domain of the alpha subunit, which closely interacts with the A domain of the beta subunit (the A domain also being known as the I-like domain of the beta subunit). The affinity state of an integrin is regulated by the conformation of the headpiece. Rearrangement of the headpiece can be initiated by intrinsic ligands and by the binding of specific adaptors to the cytoplasmic domain. The specific conformational state of an integrin receptor can have a fundamental effect on integrin function. As such, in many instances, integrin conformation is more important than integrin expression. There are three main possible conformations of the extracellular domain of integrins:

(i) a low affinity state comprising a bent conformation having a closed headpiece;

(ii) an extended conformation having a closed headpiece representing an intermediate affinity state; and (iii) a ligand-binding induced high affinity extended form having an extended open headpiece.

Current integrin antagonists fall into three classes: direct inhibitors of ligand binding to the I-domain of the alpha chain, allosteric inhibitors of the I-domain of the alpha chain and allosteric antagonists of alpha chain/beta I-like domains.

Functional modification of beta 1 integrin comprising activation and blocking of adhesion to a substrate under a defined set of conditions has been reported. Such means of modulating beta 1 integrin has certain limitations. Firstly, modulation does not take into account that integrins can exist in (i) inactive, (ii) active and (iii) active and occupied states. Secondly, functional modulation is often achieved via different domains and, as each region appears to possess a different function, may entail different downstream intracellular signalling therefore even if the effect on adhesion is similar, the functional end outcome can be different. Further, beta 1 integrin is known to exist in four different splice variants, with the resulting differences in the cytoplasmic domain implicating different downstream signalling.

There have been numerous publications documenting a potential use of betal integrin by a variety of enveloped and non-enveloped viruses, bacteria and parasites as primary receptor to infect host cells. Due to its ubiquitous expression and internalisation and/or recycling mechanism, it lends itself as an attractive target for host infection where the binding of pathogens to betal integrin either as a primary receptor or in subsequent internalisation process and initiates a cascade of signalling events leading to endocytosis and intracellular trafficking of the pathogen.

Direct binding has been shown in the cases of picornaviruse and hantaviruses. Reports have described the mechanism by which viruses hijack the use of integrin in infection. Principally, viruses mimic integrin ligands in their receptorbinding mechanism resulting in the same conformational changes seen in integrin activation such as integrin conformational activation, clustering and the signalling events which are required for receptor internalization.

Secondary binding to betal integrin leading to internalisation has been described for certain adenoviruses, reoviruses and several herpesviruses.

This has led the inventors to test whether modification of betal integrin conformation and preventing its activation could have an effect on Ebola virus (EBOV) infection. The activity/conformation state, and hence binding affinity, of integrins on the surface of cells can alter specific intracellular signalling pathways leading to alterations in cell adherence and receptor internalisation and recycling.

EBOV has caused few outbreaks in the African continent in recent years. The EBOV outbreak in 2014 in West Africa has been described by experts to the largest and most complicated that the world has ever seen. In less than six months, EBOV has killed more than 1,900 people in four African nations; Guinea, Liberia, Sierra Leone, and Nigeria. The WHO has indicated that the number of cases could have been underestimated.

EBOV virus is one of a group of zoonotic viruses that can cause severe viral haemorrhagic fever and is associated with high death rate (50-80%). Such viruses are of particular public health importance because of their ability to spread to carers and healthcare workers, difficulties in rapid detection, and the lack of treatments.

To date there are no licensed cures or vaccines available. Therefore, the best way to case management are early recognition and isolation of cases, use of personal protective equipment, and supportive medical care to reduce mortality. However, a small number of biotechnology companies (Sarepta have an experimental treatment which has not yet been clinically tested). One such vaccine is the experimental antibody Zmapp. To date, no drug has been developed in such infectious diseases where host mechanisms are the primary target.

EBOV is an enveloped, negative-stranded RNA virus It is a member of the family of Filoviridae. EBOV infection in human causes Ebola viral hemorrhagic fever. To date, five subtypes of EBOV have been identified

a. Zaire,

b. Sudan,

c. Cote d’Ivoire,

d. Reston and

e. Bundibugyo.

Of all the subtypes, EBOV Zaire is the most pathogenic in humans, with mortality rates reaching 90%. The 2014 epidemic is caused by the Zaire strain of Ebola virus.

EBOV is known to encode two forms of its glycoprotein.

a. a dimeric, secreted form (sGP) transcribed from the viral RNA with an unknown function and

b. A second type of glycoprotein which results from transcriptional editing of the glycoprotein ORF and encodes a trimeric, membrane-bound form (GP). This form is expressed at the cell surface and is incorporated into the virion and drives viral attachment and membrane fusion.

GP is translated as a precursor then cleaved by furin in the Golgi into two covalently connected subunits, a surface subunit, GP1 and a membrane-spanning subunit, GP2. As observed in other viral infections,

Although, the cause of the high pathogenicity of EBOV is not well understood, immune dysregulation has been shown to play a role. In addition, EBOV GP has appears to modulate the expression of host surface proteins involved in cellular recognition and entry, most notably major are integrins.

Indeed, expression of high levels of EBOV GP in cultured cells was reported to disrupt cell adhesion resulting in loss of cell-cell contacts as well as cell rounding and loss of attachment to the culture substrate leading to anoikis.

Betal integrins have been proposed to facilitate entry of the highly virulent filovirus, EBOV. Cells transfected with the EBOV GP have been shown to have what appears to be reduced expression of that betal integrin. This apparent reduction in betal integrin expression could also be attributed to increased recycling secondary to conformational activation. More recently, Francica et al.

have demonstrated that EBOV impedes recognition of betal integrin by steric occlusion on the cell surface.

However, Takada et al. hypothesized that GP interacts with betal integrins, leading to its down-regulation. In this study, Takada et al. supported their hypothesis by testing the effect of treatment of target cells with antibodies against betal integrin hybrid domain using JBla clone. JBla treatment reduced GP-pseudotyped virus infection by 50%. Subsequent studies demonstrated that rather than being needed for virus binding or internalization, betal integrin was required for steps leading to fusion. Such steps involve the protease cathepsin and Neimann Pick protein Cl (NPC1). The latter functions in similar fashion to the enzyme sphingomyelinase and is essential for EBOV infection. The inventors have previously shown that allosteric modulation of betal integrin inhibited the injury-induced sphingomyelinase activity and alteration in cell membrane composition.

As the inventors hypothesised a link between EBOV, betal integrin and sphingolipids, we also sought to look into identifying how to enhance anti-viral efficacy against EBOV infection. We identified Ezetimibe which is a drug that lowers plasma cholesterol levels. It is marketed by MSD as Zetia or Ezetrol or its derivatives. It does so by targeting NPC1L1, a receptor which has ~50% homology with NPC1, and is an approved and generally safe drug used for the management of hypercholesterolemia. We hypothesised therefore that NPC1 can be blocked with Ezetimibe or its derivatives. Recently, there have been reports on its efficacy against hepatitis viral infection and tuberculosis.

Summary of the Invention

According to a first aspect of the present invention there is provided a method for inhibiting and/or treating EBOV infection and effects, the method comprising a step of administering a therapeutically effective amount of Ezetimibe or its derivatives alone or in combination with a compound which mediates allosteric modulation of beta 1 integrin to a subject in need thereof.

The compounds may be administered simultaneously, sequentially or separately to EBOV affected patients or healthy subjects. The compounds may be administered prior to exposure EBOV.

According to a second aspect of the present invention there is provided a treatment combination consisting of a compound which mediates allosteric modulation of beta 1 integrin and Ezetimibe or its derivatives for use in inhibiting and/or treating EBOV infection and effects.

In certain embodiments, the compounds are administered concurrently with or following the EBOV exposure.

Treatment using the above method improves the condition of patients as compared to subjects whom have not been treated.

The therapeutically effective amount” of the compound as referred to above is an amount which is sufficient to result in the allosteric modulation of beta 1 integrin and inhibition of the sphingomylinases such as NPC1 using Ezetimibe or its derivatives.

Typically, the allosteric modulation of beta 1 integrin results in beta 1 integrin assuming a specific intermediate affinity state conformation wherein the extracellular domain comprises an extended conformation having a closed headpiece (partial stimulatory effect). The methods of the invention therefore comprise a step of administering a compound which mediates allosteric modulation of beta 1 integrin (an allosteric modulator compound) into the intermediate affinity state conformation. The methods of the present invention comprise a step of exposing beta 1 integrin to a therapeutically effective amount of the allosteric modulator compound, wherein said allosteric modulator binds to beta 1 integrin and mediates an allosteric change such that beta 1 integrin assumes an intermediate affinity state conformation with partial stimulatory effect.

The compound which the mediates allosteric modulation of beta 1 integrin should be capable of binding to beta 1 integrin regardless of the structural conformation of beta 1 integrin, that is, that the compound binds to beta 1 integrin irrespective of whether beta 1 integrin is in a low or high affinity conformation state. Accordingly, in certain embodiments the compound which mediates allosteric modulation of beta 1 integrin induces a conformational change from a high affinity conformational state, wherein the extracellular domain is in an extended form with an open headpiece to the intermediate partially stimulated affinity state conformation. In an alternative embodiment, the compound which mediates allosteric modulation of beta 1 integrin induces a conformational change from an inactive conformational state, where the extracellular domain is in a closed bent conformation, to the intermediate partially stimulated affinity state conformation.

In certain embodiments, the allosteric modulation of beta 1 integrin into the intermediate affinity state conformation in combination with Ezetimibe or its derivatives is monitored by monitoring for EBOV titer or viraemia.

Typically, the compound which mediates the allosteric modulation of beta 1 integrin binds to beta 1 integrin

Typically, the combination includes a compound which mediates the allosteric modulation of betal integrin and a compound which inhibits the function of the sphingomyelinase NPC1 such as Ezetimibe or its derivatives.

In certain embodiments, the compound binds to at least one epitope present on the extracellular domain of beta 1 integrin. Typically, the epitope which is bound by the compound is distinct to the ligand binding epitope of beta 1 integrin. Typically, the compound binds to the hybrid domain of beta 1 integrin.

In certain embodiments, the antibody is the HUTS21 clone or a humanised version or fragment thereof. In certain embodiments, the antibody is the B44 clone or a humanised version or fragment thereof.

The allosteric modulator compound may be a disintegrin, or a variant or an analogue thereof.

In certain embodiments, the compound which mediates the allosteric modulation of beta 1 integrin binds to beta 1 integrin within the second hybrid domain, e.g. in the region of amino acid residues 355 to 425 of mature human beta 1 integrin.

In certain embodiments, the compound which mediates allosteric modulation of beta 1 integrin is selected from the group consisting of a peptide, an antibody or antigen binding fragment thereof, a small molecule (low molecular weight), a peptidomimetic, a nucleic acid, a polynucleotide, a polysaccharide, an oligopeptide, a carbohydrate, a lipid, an aptamer, a naturally occurring compound such as a plant derived compound, a chemical, non-antibody modulating agents which are distinct from oligopeptide fragments of integrin ligands (e.g., ECM proteins, such as fibrinogen and fibronectin) and cyclic derivativess of these fragments. In certain embodiments, the compound which mediates allosteric modulation of beta 1 integrin is selected from the group consisting of a compound derived from a fibronectin protein, for example the CBD portion of fibronectin or the RGD sequence of the CBD; a vitronectin, or an analogue of an integrin binding portion of vitronectin; a laminin, or an analogue or an integrin binding portion of a laminin; a collagen, or an analogue or an integrin binding portion of a collagen; a polypeptide other than an ECM molecule which binds to a beta 1 chain of integrin, e.g., a CBD-binding portion of integrin; a polypeptide selected for binding in, for example, a phage display or a 2-hybrid assay; and a small molecule, e.g., a small molecule capable of binding a beta 1 chain of integrin, such as a CBD-binding portion of integrin. In certain embodiments, the compound comprises a nucleic acid (polynucleotide) which encodes one of the above-described compounds.

In certain embodiments, the compound which mediates allosteric modulation of beta 1 integrin is an antibody or an antigen binding fragment thereof having binding specificity for beta 1 integrin, wherein binding of said antibody or fragment mediates a conformational change which induces beta 1 integrin to assume the intermediate affinity state conformation. Typically, the antibody or fragment binds to the hybrid domain of beta 1 integrin, e.g. within the region of amino acid residues 355-425 of mature beta 1 integrin. The antibody may be a chimeric antibody, a humanised antibody or a monoclonal antibody. In certain embodiments, the antibody may bind to an epitope on beta 1 integrin with a dissociation constant (Kd) selected from the group of from about 10 ⁷M to about 10 ⁿM.

In certain embodiments, the antibody is produced by a commercial clone, B44 HUTS4 or HUTS21, or is a humanised version thereof. The B44 monoclonal antibody is obtainable as a commercial clone B44 from Millipore (MAB2259Z, Millipore Europe Ltd, Hampshire, UK). The HUTS4 monoclonal antibody is obtainable as a commercial clone from Millipore (MAB2079Z). The HUTS21 monoclonal antibody is obtainable as a commercial clone HUTS21 from BD Biosciences (Cat #556047). B44, HUTS4 and HUTS21 bind to an epitope within the region of amino acid residues 355-425 of the beta 1 integrin hybrid domain irrespective of the conformational state of the receptor. In certain embodiments, the compound which mediates allosteric modulation of beta 1 integrin is a binding fragment derived from the B44 monoclonal antibody or a humanised version thereof. For example, the compound may be a F’ab fragment, a scFV fragment or a fusion protein which is derived from, and maintains the binding specificity of, the B44 monoclonal antibody.

In certain embodiments, the subject is a mammal, typically a human. In certain embodiments, the subject is suffering from, or at risk of developing, EBOV. In certain embodiments, the subject has been, or is, exposed to EBOV..

According to a further aspect of the present invention there is provided a method for identifying compounds for use in the inhibition and/or treatment of EBOV and associated conditions and effects, the method comprising a step of screening candidate compounds for the ability to allosterically modify beta 1 integrin, and a compound which affect sphingomyelinase function, wherein the combination of allosteric modulation of beta 1 integrin and inhibition of sphingomyelinase by candidate compound combination is indicative of utility in the inhibition and/or treatment of EBOV infection and associated conditions and effects.

Typically, the allosteric modulation of beta 1 integrin results in beta 1 integrin assuming an intermediate affinity partially activated state conformation. The method therefore comprises a step of screening candidate compounds for the ability to allosterically modify beta 1 integrin into the intermediate partially activated affinity state conformation, wherein allosteric modulation of beta 1 integrin by a candidate compound into the intermediate partially activated affinity state conformation is indicative of utility of that compound in the inhibition and/or treatment of EBOV infection and associated conditions and effects. In certain embodiments, the intermediate partially activated affinity state conformation is assayed for using FRET data.

In certain embodiments, candidate compounds for screening may be selected from compounds known to bind beta 1 integrin and/or inhibition of sphingomyelinases such as NPC1.

The assay method of the invention may be carried out in vitro.

A further aspect of the present invention provides compounds identified by the screening method of the invention and use thereof in the inhibition and/or treatment of EBOV infection and association conditions and effects.

As defined herein, the term allosteric modulator compound” or similar relates to a compound which can allosterically modulate a protein, in this case beta 1 integrin. Allosteric modulation refers to a control mechanism which alters protein behaviour wherein binding of the allosteric modulator compound occurs at a site distinct from the active site, this resulting in a conformational change which influences protein function.

As herein defined, the term intermediate affinity state conformation” describes the conformation of the extracellular domain of beta 1 integrin when under normal homeostatic conditions, that is, when beta 1 integrin is in a conformational equilibrium between the accepted high and low affinity states. The intermediate partially activated affinity state conformation can be differentiated from the low and high affinity states which beta 1 integrin is known to assume following inactivation and insult/injury respectively. In the intermediate partially activated affinity state conformation, beta 1 integrin is not in a completely inactive state. However, a complete swing” out of the hybrid domain, a process required in order for beta 1 integrin to assume a high affinity conformation, is inhibited. Further, the intermediate partially activated affinity state conformation can be further characterised in that it maintains extracellular binding. The change in conformation in the extracellular domain of beta 1 integrin is mediated by the opening or closing of the hinge angle between the A domain and the hybrid domain. The opening of this hinge is indicative of a conformational switch to a high binding affinity. Inactive beta 1 integrin has a bent conformation, wherein the headpiece is spaced approximately 5 nm from the cell membrane upon which the integrin is expressed. In the activated, extended conformation, the headpiece projects from the membrane at a distance of between 20 to 25 nm. In the intermediate affinity state conformation, where the extracellular domain of the integrin is extended, but the headpiece is in a closed conformation, the headpiece can be between about 6 nm to about 19 nm from the cell membrane.

Disintegrins are a family of naturally-occurring cysteine-rich peptides originally isolated from viper venom, but also found on cells and elsewhere, many of which contain the sequence Arg Gly Asp (RGD) as an integrin recognition site. Disintegrins are defined by their specific amino acid sequences and threedimensional structures. Variants of disintegrins are disintegrins engineered to have one or more amino acids added, deleted or replaced. Analogues are nonpeptide mimetics of disintegrins or their variants.

Antibodies

An antibody” is an immunoglobulin, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses and fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd, and a bispecific antibody. So called domain antibodies” may also be produced, as can binding fragments based on or derived from Camelid or shark antibodies.

As antibodies can be modified in a number of ways, the term antibody” should be construed as covering any binding member or substance having a binding domain with the required specificity. The antibody of the invention may be a monoclonal antibody, or a fragment, derivative, functional equivalent or homologue thereof. The term includes any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.

The constant region of the antibody may be of any suitable immunoglobulin subtype. However, it is preferred that the antibody subtype is IgGl. However, in alternative embodiments, the subtype of the antibody may be of the class IgA, IgM, IgD and IgE where a human immunoglobulin molecule is used. Such an antibody may further belong to any sub class, e.g. IgGl, IgG2a, 2b, IgG3 and IgG4. In further embodiments, the constant region may be derived from an immunoglobulin subtype from a non-human source such as any other animal, in particular a mouse.

Fragments of a whole antibody can perform the function of antigen binding. Examples of such binding fragments are: a Fab fragment comprising the VL, VH, CL and CHI antibody domains; an Fv fragment consisting of the VL and VH domains of a single antibody; a F(ab’)2 fragment; a bivalent fragment comprising two linked Fab fragments; a single chain Fv molecule (scFv) wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; or a bi-specific antibody, which may be multivalent or multispecific fragments constructed by gene fusion.

A fragment of an antibody or of a polypeptide for use in the present invention, for example, a fragment of the B44 antibody, generally means a stretch of amino acid residues of at least 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, more preferably at least about 20 to 30 or more contiguous amino acids and most preferably at least about 30 to 40 or more consecutive amino acids. A preferred group of fragments are those which include all or part of the CDR regions of monoclonal antibody B44.

A derivative” of such an antibody or polypeptide, or of a fragment of a B44 antibody, means an antibody or polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids, preferably while providing a peptide having beta 1 integrin binding activity. Preferably such derivatives involve the insertion, addition, deletion and/or substitution of 25 or fewer amino acids, more preferably of 15 or fewer, even more preferably of 10 or fewer, more preferably still of 4 or fewer and most preferably of 1 or 2 amino acids only.

The term antibody includes antibodies which have been humanised. Methods for making humanised antibodies are known in the art. A humanised antibody may be a modified antibody having the hypervariable region of a monoclonal antibody such as B44 and the constant region of a human antibody. Thus, the binding member may comprise a human constant region.

The variable region other than the hypervariable region may also be derived from the variable region of a human antibody and/or may also be derived from a monoclonal antibody such as B44. In such cases, the entire variable region may be derived from murine monoclonal antibody B44 and the antibody is said to be chimerised. Methods for making chimerised antibodies are known in the art.

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of the antibodies produced.

As used herein, reference to “B44” includes humanised versions thereof and sequences which show substantial homology with B44 and which can be characterised in that they exhibit substantially identical binding specificity to that defined herein in relation to B44. Preferably the degree of homology between B44 complementarity determining regions (CDRs) and the CDRs of other antibodies will be at least 60%, more preferably 70%, further preferably 80%, even more preferably 90% or most preferably 95%.

Treatment / Therapy

The term 'treatment’ is used herein to refer to any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (inhibitative treatment). Treatment may include curative, alleviation or prophylactic effects. It also includes cosmetic preparations.

More specifically, reference herein to therapeutic and inhibition” or prophylactic treatment is to be considered in its broadest context. The term therapeutic does not necessarily imply that a subject is treated until total recovery. Similarly, prophylactic does not necessarily mean that the subject will not eventually contract a disease condition. References to inhibiting” or inhibition” are intended to encompass partially inhibiting or partial inhibition. Similarly, references to reversing” or reversal” are intended to encompass partially reversing or partial reversal.

Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or inhibiting or otherwise reducing the risk of developing a particular condition. The term prophylactic may be considered as reducing the severity or the onset of a particular condition. Therapeutic may also reduce the severity of an existing condition.

Administration

The allosteric modulatory compound and Ezetimibe or its derivatives of the present invention, such as an antibody, small chemical entity or protein, may be administered alone, but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration. The allosteric modulator compound and Ezetimibe and its derivatives may be administered to a patient in need of treatment via any suitable route.

Routes of administration may include parenteral administration, including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch. Further suitable routes of administration include, but are not limited to, oral, rectal, nasal, topical, including buccal and sublingual, infusion, vaginal, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration or administration via oral or nasal inhalation, by means of, for example, a nebuliser or inhaler, or by an implant.

The compound or pharmaceutical composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semi-permeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules.

Mimetics

A substance identified as an allosteric modulator of beta 1 integrin may be peptide or non-peptide in nature. Non-peptide small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly, a peptide) may be designed for pharmaceutical uses. The designing of mimetics to a known pharmaceutically active compound is a known approach in the development of pharmaceuticals based on a lead” compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening a large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its pharmacophore”.

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Use of mimetics of substances identified as having the ability to modulate the conformation of beta 1 integrin and Ezetimibe or its derivatives are included within the scope of the present invention. A polypeptide, peptide or substance which can modulate the conformation of beta 1 integrin according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment. Such a kit may include instructions for use.

Peptidomimetics

Whilst numerous strategies to improve the pharmaceutical properties of peptides found to exert biological effects are known in the art including, for example, amide bond replacements, incorporation of non-peptide moieties, peptide small molecule conjugates or backbone cyclisation, the optimisation of pharmacological properties for particular peptides still presents those involved in the optimisation of such pharmaceutical agents with considerable challenges.

Peptides for use in the present invention may be modified such that they comprise amide bond replacement, incorporation of non peptide moieties or backbone cyclisation. Suitably if cysteine is present, the thiol of this residue is capped to inhibit damage of the free sulphate group. Suitably a peptide for use in the present invention may be modified from the natural sequence to protect the peptides from protease attack.

Suitably a peptide for use in the present invention may be further modified using at least one of C and / or N-terminal capping, and / or cysteine residue capping. Suitably a peptide for use in the present invention may be capped at the N terminal residue with an acetyl group. Suitably a peptide for use in the present invention may be capped at the C terminal with an amide group. Suitably the thiol groups of cysteines are capped with acetamido methyl groups.

Pharmaceutical Compositions

Pharmaceutical compositions for use in accordance with the present invention may comprise, in addition to the active ingredient (i.e. an allosteric modulator of beta 1 integrin which induces the beta 1 integrin to assume an intermediate affinity state and Ezetimibe or its derivatives), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation.

Dose

The compounds of the present invention are preferably administered to an individual in a therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and timecourse of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners, physicians or other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.

Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Throughout the specification, unless the context demands otherwise, the terms comprise” or include”, or variations such as comprises” or comprising”, includes” or including” will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

The present invention will now be described with reference to the following examples, which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention.

EXAMPLE 1

Ezetimibe and derivatives (Ezetimibe, Ezetimibe-O-b-Glucoronide Benzylic (Ezetimibe GB) and Ezetimibe-O-b-Glucoronide Phenolic (Ezetimibe GPh) and two antibodies (JBla and B44) were tested in an EBOV infection assay with the HeLa and HFF cell lines.

Stock solutions of compounds were made at ~10mM concentration in 100% DMSO.

Antibody were received JB1 at 0.5mg/ml and B44 at 1.5mg/ml

Compounds and antibody (AB) activity was tested in a 10-point dose response with 2 fold step dilution (see table below). The highest concentration was ΙΟΟμΜ (final). All doses were repeated 4 times on a single plate (n=4). DMSO stocks were tittered in DMSO and then equal volumes of each dose were transferred into Media. All assay wells had 1% DMSO final.

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infected wells treated with 1% DMSO were used as a neutral control. Additionally, 16 wells were not infected and were used as a low signal controls.

Cells were infected with EBOV(Zaire) at M01=0.5. Infection was stopped after 48h by fixing cells with a formalin solution.

To detect infected cells, an immuno-staining was completed with anti-GP antibodies.

Images were taken by the PE Opera confocal platform using a lOx objective and were analysed using Acapella software. Signal for GP-staining was converted into % infection.

The number of nuclei per well was used to determine % viability of cells (in comparison to infected but untreated controls, n=16).

Data was analysed using GeneData software. % of Infection was converted into % Inhibition (%INH) for each well using plate controls.

Results summary

Both Ezetimibe and Ezetimibe-O-b-Glucoronide Benzylic (Ezetimibe GB) showed inhibitory effect on EBOV (Figure 1). However, Ezetimibe and Ezetimibe-O-bGlucoronide Phenolic (Ezetimibe GPh) , the latter showing no anti-viral effect, had significant cytotoxicity at the effective anti-EBOV concentrations.

Beta 1 integrin targeting using JBla had no effect on EBOV at any concentration tested (Figure 2). However, targeting betal integrin using B44, which targets another part of the hybrid domain and affect conformation, resulted in upto 40% inhibition of EBOV (Figure 2).

EXAMPLE 2

Experiments were repeated as described in Example 1 but with altering the concentrations of ezetimibe and its derivatives and testing them in combination with anti-betal integrin antibodies (B44). The study layout is detailed in Figure 5 3.

Results Summary

The inventors have found that the combination of B44 + Ezetimibe achieved up to ~70% inhibition of EBOV (Figure 4). The effect was additive with no clear synergy observed at the tested concentrations.

Ezetimibe shows toxicity >50% at concentrations > 20 uM (Figure 4)

However, surprisingly there was synergistic effect of B44 + EzetimibeGB with inhibitory efficacy of 80% against EBOV (Figure 5). Synergy was observed for

B44 at the following concentrations 20 ng/ml, 60ng/ml, 190ng/ml, 500ng/ml and 1.7ug/ml (Figure 6). The effect at the highest and lowest doses was additive (Figure 7). EzetimibeGB and B44 showed no toxicity at any of the tested concentrations (Figure 5).

10 17

Methods for the treatment and prevention of Ebola

Claims

1. A method for inhibiting and/or treating EBOV infection and effects comprising a step of administering a therapeutically effective amount of

5 Ezetimibe or its derivatives alone or in combination with a compound which mediates allosteric modulation of beta 1 integrin to a subject in need thereof.

2. The method as claimed in claim 1 wherein the compounds may be administered simultaneously, sequentially or separately to EBOV 10 affected patients

3. The method as claimed in claim 1 wherein the compound may be administered simultaneously, sequentially or separately to healthy subjects prior to exposure EBOV.

4. A treatment combination consisting of a compound which mediates

15 allosteric modulation of beta 1 integrin and Ezetimibe or its derivatives for use in inhibiting and/or treating EBOV infection and effects.

5. The treatment claimed in claim 4 wherein the compounds are administered concurrently with or following the EBOV exposure.

6. The therapeutically effective amount” of the compound as referred to in

20 claims 1 and 4 is an amount which is sufficient to result in the allosteric modulation of beta 1 integrin and inhibition of the sphingomylinases such as NPC1 using Ezetimibe or its derivatives.

7. The allosteric modulation of beta 1 integrin as claimed in claims 1 and 4 results in beta 1 integrin assuming a specific intermediate affinity state 25 conformation wherein the extracellular domain comprises an extended conformation having a closed headpiece (partial stimulatory effect).

8. A compound which the mediates allosteric modulation of beta 1 integrin capable of binding to beta 1 integrin regardless of the structural conformation of beta 1 integrin, that is, that the compound binds to beta 30 1 integrin irrespective of whether beta 1 integrin is in a low or high affinity conformation state.

19 10 17

9. A method wherein the allosteric modulation of beta 1 integrin into the intermediate affinity state conformation in combination with Ezetimibe or its derivatives is monitored by monitoring for EBOV titer or viraemia.

10. The combination as claimed in claim 9 includes a compound which mediates the allosteric modulation of betal integrin and a compound which inhibits the function of the sphingomyelinase NPC1 such as Ezetimibe or its derivatives.

11. A Compound which binds to at least one epitope present on the extracellular domain of beta 1 integrin.

12. A method wherein any of the preceding claims the epitope which is bound by the compound is distinct to the ligand binding epitope of beta 1 integrin.

13. A method wherein any of the preceding claims binds to the hybrid domain of beta 1 integrin.

14. A method wherein the compound in any of the preceding claims is an antibody.

15. A compound as claimed in any one of claims wherein the compound is a humanised or chimaeric antibody.

16. The antibody as claimed in claim 14 is the HUTS21 clone or a derivatives as claimed in claim 15 or fragment thereof.

17. The antibody as claimed in claim 14 is the B44 clone or a derivatives as claimed in claim 15 or fragment thereof.

18. A betal integrin allosteric modulator compound as claimed by any of the preceding claims may be a disintegrin, or a variant or an analogue thereof.

19. A compound which mediates allosteric modulation of beta 1 integrin as in preceding claims is selected from the group consisting of a peptide, an antibody or antigen binding fragment thereof, a small molecule (low molecular weight), a peptidomimetic, a nucleic acid, a polynucleotide, a polysaccharide, an oligopeptide, a carbohydrate, a lipid, an aptamer, a naturally occurring compound such as a plant derived compound, a chemical, non-antibody modulating agents which are distinct from

19 10 17 oligopeptide fragments of integrin ligands (e.g., ECM proteins, such as fibrinogen and fibronectin) and cyclic derivativess of these fragments.

20. A compound which mediates allosteric modulation of beta 1 integrin as in preceding claims is selected from the group consisting of a compound derived from a fibronectin protein, for example the CBD portion of fibronectin or the RGD sequence of the CBD; a vitronectin, or an analogue of an integrin binding portion of vitronectin; a laminin, or an analogue or an integrin binding portion of a laminin; a collagen, or an analogue or an integrin binding portion of a collagen; a polypeptide other than an ECM molecule which binds to a beta 1 chain of integrin, e.g., a CBD-binding portion of integrin; a polypeptide selected for binding in, for example, a phage display or a 2-hybrid assay; and a small molecule, e.g., a small molecule capable of binding a beta 1 chain of integrin, such as a CBD-binding portion of integrin.

21. A compound which mediates allosteric modulation of beta 1 integrin as in preceding claims comprises a nucleic acid (polynucleotide) which encodes one of the above-described compounds.

22. A compound which mediates allosteric modulation of beta 1 integrin as in the preceding claims is an antibody, a chimeric antibody, a humanised antibody, monoclonal antibody or an antigen binding fragment thereof having binding specificity for beta 1 integrin, wherein binding of said antibody or fragment mediates a conformational change which induces beta 1 integrin to assume the intermediate affinity state conformation.

23. An antibody or fragment as in claim 22 binds to the hybrid domain of beta 1 integrin.

24. A method for treatment using compound which mediates allosteric modulation of betal integrin as in the preceding claims to be administered to the subject wherein the subject is a mammal, typically a human suffering from, or at risk of developing, EBOV.

25. An assay method for identifying compounds for use in the inhibition and/or treatment of EBOV and associated conditions and effects, the

9 10 17 method comprising a step of screening candidate compounds for the ability to allosterically modify beta 1 integrin, and a compound which affect sphingomyelinase function, wherein the combination of allosteric modulation of beta 1 integrin and inhibition of sphingomyelinase by 5 candidate compound combination is indicative of utility in the inhibition and/or treatment of EBOV infection and associated conditions and effects.

26. An assay method as claimed in claim 25 wherein the allosteric modulation of beta 1 integrin results in beta 1 integrin assuming an

10 intermediate affinity partially activated state conformation is indicative of utility of that compound in the inhibition and/or treatment of EBOV infection and associated conditions and effects.

27. An assay method as claimed in claim 25 wherein the candidate compounds for screening may be selected from compounds known to 15 bind beta 1 integrin and/or inhibition of sphingomyelinases such as

NPC1.

Intellectual

Property Office

Application No: GB1617715.6 Examiner: Dr Jeremy Kaye