WO2010086617A2

WO2010086617A2 - Treatment

Info

Publication number: WO2010086617A2
Application number: PCT/GB2010/000153
Authority: WO
Inventors: Caroline Ann Rowland; Margaret Gillian Hartley; James Edward Eyles
Original assignee: The Secretary Of State For Defence
Priority date: 2009-01-29
Filing date: 2010-01-29
Publication date: 2010-08-05
Also published as: GB0901411D0; GB201001521D0; GB2467437A; WO2010086617A3

Abstract

The present invention relates to a phosphoantigen for use as a medicament, particularly in the prophylaxis and/or treatment of infection by Francisella, e.g. Francisella tularensis: and a pharmaceutical composition comprising such phosphoantigens.

Description

Treatment

The present disclosure relates to phosphoantigens for use in the treatment and/or prophylactic protection against tularemia and pharmaceutical compositions comprising said phosphoantigen for the same.

Francisella tularensis is a small pleomorphic Gram negative coccobacillus which causes the disease tularemia. Tularemia is a zoonotic infection. It circulates in populations of rodents and lagomorphs, and outbreaks in humans often parallel outbreaks in animal populations. However, it is not clear whether these animal species are the true reservoir of the bacterium in the environment. A wide range of arthropod vectors have been implicated in the transmission of tularemia between mammalian hosts including mosquitoes, ticks, and deer flies. These vectors can also transmit the pathogen to man. F. tularensis can also be acquired by contact with, or ingestion of. contaminated material including food and water, and by inhalation of infectious particles. Rural populations and especially those individuals who spend periods of lime in endemic areas such as farmers, hunters, walkers and forest workers are most at risk of contracting tularaemia. Outbreaks associated with contaminated water supplies can involve large numbers of cases, but usually the incidence of the disease is low. These water-associated outbreaks arc mainly caused by subspecies holarctica; subspecies tularensis has never been linked to water-borne infections.

The type and severity of disease is dependent on strain, dose and route of infection. F. tularensis subspecies tularensis and holarctica cause the majority of reported cases, with subspecies tularensis causing the more severe disease of the two. Although tularemia can be a severely debilitating or even fatal disease, especially when caused by F. tularensis subspecies tularensis, many cases of disease caused by lower virulence strains are undiagnosed due to the non-specific nature of the symptoms. The incubation period is 3-5 days normally (range 1 -21 ), and patients develop flu-like symptoms which may be protracted and relapsing if untreated.

Infection through the skin results in ulccroglandular tularemia. This is the most common presentation of the disease and can arise following the bile of an infected vector or through direct contact with the flesh of infected animal. Less commonly, infection can occur through the conjunctiva. This is termed oculoglandular tularemia and arises following direct contamination of the eye. Ingestion of infected meat or water can result in oropharyngeal or gastrointestinal tularemia. Inhalation of F. tularensis results in respiratory or pneumonic tularemia. Respiratory tularemia has been reported in farmers following activities such as making hay, where infectious dusts can be generated. Other high-risk activities in endemic areas are lawn-mowing and bush cutting. For example in Martha's Vineyard, Massachusetts, the majority of investigated cases were respiratory, in landscapes undertaking these types of work. Pneumonia can also arise following hacmatogcnous spread in other forms of tularaemia. Symptoms can be variable and depend on the virulence of the strain involved. Infection with the most highly virulent strains can have a case fatality rate of up to 30% if untreated, but antibiotic therapy has reduced this to around 2%. Presentation can range from a mild pneumonia to an acute infection with high fever, malaise, chills, cough, delirium and pulse-temperature dissociation. Radiological examination may reveal parenchymal infiltrates, most commonly in one lobe, and hilar lymphadenopathy may be present. The infectious dose required to cause disease by this route is very low.

Tularemia responds well to antibiotic therapy. As described above, the mortality rate of the more acute forms of the disease is reduced significantly if the patient receives suitable antibiotics. Historically aminoglycosides have been drugs of choice. Although cl inically effective, streptomycin is rarely used now due to problems of ototoxicity and nephrotoxicity. Similarly, although chloramphenicol has been used historically for treatment, it would be unlikely to be used as a first choice due to the possibil ity of irreversible effects on haematopoiesis. Gentamicin is a suitable alternative aminoglycoside, and has been used for treatment of pneumonic tularemia. Due to the requirement for parenteral dosing and monitoring of serum levels, aminoglycosides are now only used for the most serious cases. The tetracyclines have been associated with high relapse rates on withdrawal. Doxycycline is effective in treatment of tularemia, but should be avoided for use in young children due to possible effects on developing teeth. Ciprofloxacin has been shown to be highly effective in oral therapy of tularemia, and can be considered the current drug of choice for uncomplicated tularemia. It has shown to be effective in treating tularemia in children, and may be suitable for use in pregnant women.

No licensed vaccine is available for prophylaxis of tularaemia. A live vaccine strain (LVS) was developed in the 1950s, and used extensively to vaccinate at-risk workers under Investigational New Drug Status which resulted in a significant decrease in laboratory acquired infections. Although LVS appears to be effective, there have been problems with the strain, such as reversion to virulence, mixed colony morphology and variable immiinogenicity, and thus the LVS strain has failed to achieve licensing for human use.

Given the debilitating nature of tularemia that no prophylactic treatment/vaccine is available which is suitable for use in the general publ ic and the fact that the disease in some instances is difficult to treat, an effective prophylactic therapy and/or treatment of the disease would be useful.

The present disclosure provides a phosphoanligen for the prophylaxis and/or treatment of tularemia.

Whilst phosphoantigens have been suggested previously as potential adjuvants, there has been no suggestion that such compounds can be used in the treatment of a patient in their own right, that is, it has not been previously suggested that a treatment against any of the pathogens described herein could involve a phosphoantigen in the absence of an antigen, or if an antigen is present, that the antigen is not pharmaceutically active in the prophylaxis and/or treatment of tularemia, or it is not present in sufficient amounts to be pharmaceutically active in the prophylaxis and/or treatment of tularemia. As used herein, "adjuvant" refers to a non- antigenic substance or substantially non-antigenic substance that is used in combination with an antigen for enhancing the immune response against the antigen. The present invention is not intended to cover use of a phosphoantigen as an adjuvant in a medicament, such as a medicament against any of the diseases mentioned herein. As used herein, "treatment" can mean prophylactic treatment (i.e. pre-lieating) or treatment post-infection with a pathogen of the type disclosed herein. A treatment is considered successful if the symptoms of an infection are ameliorated or prevented. As used herein, "pharmaceutically active" means a substance that has a statistically significant measureablc effect on the material/host to which is administered. As used herein, "antigen specific to the pathogen" refers to an attenuated or avirulent strain of the pathogen or a subunit of the said pathogen that is peculiar to that particular type of pathogen.

Use of phosphoantigens for the treatment of a number of diseases, such as tumours, HIV. malaria and other viral and bacterial infections has been suggested. Clinical trials have been performed in relation to the treatment of tumours. However, there is little or no cl inical support to show any efficacy in other clinical indications.

Surprisingly it seems that treating an individual with phosphoantigcn provides beneficial therapeutic effects in relation to prevention of infection, or amelioration of symptoms after infection with /^•'. tularensis.

Thus there is provided a phosphoantigcn for use in the treatment or prophylaxis of F. tularensis including subspecies thereof such as tularensis.

In one aspect there is provided a method of treating a patient in need thereof comprising administering a therapeutically effective amount of a phosphoantigen.

In another aspect there is provided a use of a phosphoantigcn for the manufacture of a medicament for the treatment of infection with F. tularensis.

Most bacteria including F. tularensis produce phosphoantigens as intermediates of the DOXP metabolic pathway. Phosphoantigens are small molecular weight molecules with phosphorylated structures that selectively activate human or non-human primate T cells expressing Vγ9Vδ2 T cell receptor.

Phosphoantigens have been isolated from bacterial species such as TUBag l -4 from M. tuberculosis. Certain other phosphoanligens are known such as BrHPP and IHPP examples 1 and 2 respectively in US application publication No. 2004/0087555. The application relates to compounds of the following formula:

wherein X is a halogen selected from I, Br or Cl, R¹ is selected from methyl and ethyl, and Cat⁺ is a cation, and n is an integer between 2 and 20. A particularly suitable structure therein is:

OH O O

X — CH₂-C (CII₂),, — O 1' O P O — R3

Rl O^"Cat⁴ O CaI '

wherein X, R l and Cat+ is as defined above and R3 is selected from:

OH

X — CH₂-C (CH₂J₁₁

Rl

CH, — O

R l W C (CH₂),,

and

CH₂=C (CH₂J₁₁,

RJ

Below is a table of a number of phosphoantigcns from said application.

MOLECULE

Name Λbbicviation SliucUiie isopentenyl IPP CIb pyiophosphate

CH, C (CH₂J₂ — OPF

3-(chloromethyl)- CIHl'P CH₂CI

3-butanol-l-vl diphosphate CH, C (CH₂J₂ — OPF

OH

3-(bromomethyl)- BrI-IFP CH₁Br

3-bulanol-J-yl diphosphiiie CH., C (CIb)₂ — OFF

OH

3-(iodomethyl)-3- IHFF CH₂I butanol-J-yl diphosphiiie CH, C (CHx)₂ — OFF

OH

3-(bιomomelhvD- ISiHFFl CH₂Br

3-bulanol-l-yl tiiphosphate CH, C (CH₂J₂-OPPP

OH

3-(iodomethvl>3- IHFFI CI-M butanol- 1-yl lripliosphaie CH., C I ^" (CH₂J₂ — OFFP

OH α. γ di-3- Ii-BiHIP CI-I₂IiI CII₂Bi (broniomotliyl)-.³- butanol- l-\l H_.,C C (Cl-I₂): — (JFPFO -(-CII₂J₂ — C CII, iiiphosphate on OH α. γ di-3- di-IIlTP CIM Cl-M (iodomclhylJ-3- butanol-J-yl H.,C C r — -(CH₂J₂ — OPFPO — fCH₂J: — C r CH, tiiphosphate

OH OH wherein P is phosphate.

Other compounds explicitly disclosed in the case include: 3-(bromomethyl)-3-butanol-l-yl triphosphate (BrHPPP); 3-(iodomcthyl)-3-butanol-l-yl tiiphosphate (IHPPP); uridine 5'- triphosphate gamma-|3-methyl-3butene-l-yl|; alpha, beta di[3-biOmomethyl-3-butanol-l- yljdiphosphale.

An alternative phosplioantigcn is C-IPP

(3-methylbut-3-enyl pyrophosphonaie);

HIPP;

0 0

H'

0-P-O-P-OH I I

OH OH

HTϊglyIPP

HAngelylPP

A particularly potent phosphoaiitigcn is CHDMAPP:

Certain phosphoantigens are described in published patent application publication number US2008/0207568 (Innate Pharma) which describes certain phosphoantigens of the formula:

wherein

CaL+ represents one or more cations m is an integer 1 to 3. B is O, NH, CHF, CFT or CHi or any other isostcric group, W is C-R6 or N,

R7 is a C _{I -}I alky! group or any other isosteric group such as CF^,

R3, R4 and R6 are independently selected from H, Ci -; alky or any other isosteric group such as CFJ, R5 is selected from C₂.iacyl. aldehyde, a Ci--* alcohol or a C₂.-! ester, and Y=O^~Cat⁺ is as defined therein.

Any suitable phosphoantigen may be employed to for the prophylaxis or treatment of tularemia, including a synthetic and/or natural phosphoantigen. Particularly suitable phosphoantigens arc described above, such as CHDMAPP and IPP, which appear to be very suitable for the treatment of tularemia.

Prophylaxis as employed herein is intended to refer to wherein an individual has a reduced risk of infection i.e. invasion and/or multipl ication with the relevant pathogen compared to an individual who has not had any prophylactic treatment against the relevant pathogen.

In one embodiment there is a reduced risk of developing severe infection, for example caused by subspecies tularensis. Reduced risk as employed herein refers to a 20. 30. 40, 50, 60, 70, 80% or more reduced risk of developing the infection or a severe form thereof (such as developing the infection).

In one aspect the phosphoantigcns employed arc not compounds of the following formula:

X — CM₂- O — P — O Gu'

wherein

X is a halogen selected from I, Br or Cl, R¹ is selected from mcthy and ethyl, and Cat⁺ is a cation, and n is an integer between 2 and 20.

In one aspect the phosphoanligen is employed in combination with a cytokine, for example human recombinant inteiieukin-2 (IL-2) which may be co-administered or co-formulated with the phosphoantigcn.

In one embodiment, for example when a patient has been exposed to F. iitlciren.sis or inoculated by the same (or alternatively is going into an area with a high risk of infection) the phosphoantigen or formulation thereof, for example as defined herein, may be employed in combination with an antibiotic treatment, for example selected from aminoglycosides, gentamicin. chloramphenicol, doxycyclinc and ciprofloxacin, particularly ciprofloxacin.

Thus in one aspect, the disclosure provides a method of treating tularemia comprising administering a therapeutically effective amount of the phosphoantigen prophylactically or to a patient infected by F. lularensis.

Innoculation or exposure to the relevant pathogen in the environment is not prophylaxis or treatment with an antigen specific to the pathogen, as employed herein. Nor is it to be considered to be treatment or prophylaxis by administration of a pharmaceutical composition comprising an antigen specific to the pathogen.

In one embodiment the phosphoantigen is administered before infection, for example 1 . 2 or 3 days or a week or a month before exposure.

In one embodiment the phosphoanligen in given shortly after exposure, inoculation or infection, for example I hour to 3 days, such as 2 to 24 hours after.

In one embodiment the phosphoantigen is administered before exposure and shortly after exposure, for example as per the time frames above. In one embodiment lhe phosphoantigen is employed for the treatment of chronic infection of tularemia.

The in vitro data with the relevant pathogens indicates that the bacterial load in a human monocytic cell line is reduced 10 to 100 fold in the presence of human blood or purified γδ T cells treated with phosphoantigen and human recombinant IL-2 ex vivo. Given that the cells employed in the in vitro assay are retrieved from the blood of healthy individuals' significant confidence can be gained that the phosphoantigcns will work by the same mechanism in vivo.

In one embodiment the disclosure relates to a pharmaceutical composition comprising phosphoantigen in the presence or absence of IL-2, along with a pharmaceutically acceptable excipient for the treatment or prophylaxis of F. tularensis infection. The composition may be employed as described above for the phosphoantigen.

In one embodiment the phosphoantigen is employed in combination with IL-2 and/or a further cytokine such as IL- 15, IL-21 , interferon-γ and/or interferon-α.

The active components of the combinations of the disclosure may, for example, be co- formulated if they arc stable when mixed together. Alternatively, the components may be formulated separately and co-administcrcd. that is administered at the same time or approximately the same time, for example one immediately after the other. In further alternative embodiments the components may be administered sequentially, that is to say with a delay between the administration of each component, for example a delay of I to 1 2 hours.

Formulations

The phosphoantigens or compositions according to the present disclosure maybe administered orally, topically, parenterally, transdermal Iy, as a suppository or by any other pharmaceutically appropriate route.

Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or by subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal routes, intradermal and transdermal administration.

In one embodiment the phosphoantigen according to the disclosure is provided optionally in cither as a lyophilizcd formulation for rcconstitution later or as a liquid formulation.

Transdermal administration, such as by iontophoresis, may also be an effective method to deliver phosphoanligen to muscle. Epidermal administration may also be employed. Thus the disclosure also extends to delivery by a transdermal patch, which may be occlusive or non- occlusivc.

The actives can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the earner is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous or oily solutions of the active ingredient. For further discussions of nasal administration of AIDS-related vaccines, references are made to the following patents, U.S. Pat. Nos. 5,846,978, 5,663. 169. 5,578,597, 5,502,060, 5.476,874, 5,413,999, 5,308,854, 5, 192,668, and 5, 187,074.

Compositions of use in the disclosure include liquid preparations, for an orifice, e.g., oral. nasal, anal, vaginal, etc. administration, such as suspensions, syrups or elixirs: and. preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.

In compositions of the disclosure the relevant active ingredient may be in admixture with a suitable earner, diluent, or cxcipicnt such as sterile water, physiological sal ine, glucose or the like.

The active ingredients can be incorporated, if desired, into l iposomes, microspheres or other polymer matrices (Feigner el al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome

Technology, VoIs. I to III (2nd ed. 1993). each of which is incorporated herein by reference).

Liposomes, for example, which consist of phospholipids or other lipids, arc nontoxic. physiologically acceptable and mctabolizablc carriers that arc relatively simple to make and administer.

Liposome carriers may serve to target a particular tissue or infected cells, as well as increase the half-life of the active. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the vaccine to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to. e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired immunogen of the disclosure can be directed to the site of lymphoid cells, where the liposomes then deliver the immunogcn(s). Liposomes may be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of. e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in. e.g.. Szoka. et al.. Ann. Rev. Biophys. Bioeng. 9:467 ( 1980). U.S. Pat. Nos. 4,235.87 1. 4.501 .728. 4.837.028. and 5.019.369.

The liposomes generally contain a neutral lipid, for example phosphatidylchol ine, which is usually non-crystalline al room temperature, for example egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine or dilauryl phosphatidylcholine.

Optionally the formulation may comprise an adjuvant, for example a known adjuvant formulation may be used to reconstitute the formulation. Phosphoantigens employed in die disclosure may be mixed or adsorbed with adjuvants, which include but arc not limited to alum, muramyl dipcptidc and saponins such as Quil A. This may further boost the immune system's ability to deal with the infection.

Particular adjuvants are those selected from the group of metal salts, oil in water emulsions. Toll like receptors agonist, (in particular Toll like receptor 2 agonist, Toll l ike receptor 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8 agonist and Toll like receptor 9 agonist), saponins or combinations thereof. The level of free antigen in a given formulation may be increased by. for example, formulating the composition in the presence of phosphate ions, such as phosphate buffered saline, or by increasing the ratio of antigen to metal salt. In one embodiment the adjuvant does not include a metal salt as sole adjuvant. In one embodiment the adjuvant does not include a metal salt.

In an embodiment the adjuvant is a Toll like receptor (TLR) 4 ligand, for example an agonist such as a lipid A derivative, in particular monophosphoryl lipid A or more specifically 3- deacylated monophoshoryl lipid A (3D-MPL).

3-Deacylated monophosphoryl lipid A is known from US patent No. 4,912,094 and UK patent application No. 2,220.2 1 1 (Ribi) and is available from Ribi Immunochcm, Montana, USA.

3D-MPL is sold under the trademark MPL® by Corixa Corporation and primarily promotes CD4+ T cell responses with an IFN-g (Th 0 phenotype. It can be produced according to the methods disclosed in GB 22202 1 1 A. Chemically it is a mixture of 3-dcacylatcd monophosphoryl lipid A with 3, 4, 5 or 6 acylatcd chains. Generally in the compositions of the present disclosure small particle 3D-MPL is used. Small particle 3D-MPL has a particle size such that it may be sterile- filtered through a 0.22μm filter. Such preparations are described in International Patent Application No. WO 94/21292.

Synthetic derivatives of lipid A arc known and thought to be TLR 4 agonists including, but not limited to:

OM 197 MP-Ac DP ( 3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylaminoJ^-oxo-S- aza-9- [(R)-3 -hydroxytetradecanoylamino] decan- 1 , 10-diol, 1 -dihydrogenophosphate 10-(6- aminohexanoate) (WO 01 /46127).

Typically when 3D-MPL is used the antigen and 3D-MPL arc delivered with alum or presented in an oil in water emulsion or multiple oil in water emulsions. The incorporation of 3D-MPL is advantageous since it is a stimulator of effector T-ccll responses. Alternatively the 3D-MPL may be formulated as liposomes. Other TLR4 ligands which may be used are alkyl GIucosaminide phosphates (AGPs) such as those disclosed in WO 98/50399 or US 6303347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in LJS 6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are thought to be useful as adjuvants.

Another immunoslimulant for use in the present disclosure is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quilaja Saponaria Molina and was first described as having adjuvant activity by Dalsgaard ct al. in 1974 ("Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243- 254). Purified fragments of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS2 J (also known as QA7 and QA21 ). QS2 I is a natural saponin derived from the bark of Quillaja saponaria Molina which induces CD8+ cytotoxic T cells (CTLs), Thi cells and a predominant IgG2a antibody response.

Particular formulations of QS2 I have been described which further comprise a sterol (WO 96/33739). The ratio of QS21 : sterol will typically be in the order of 1 : 100 to 1 : 1 weight to weight.

Generally an excess of sterol is present, the ratio of QS2 1 :stcrol being at least 1 :2 w/w. Typically for human administration QS2 I and sterol will be present in a vaccine in the range of about 1 μg to about 100 μg. such as about I O μg to about 50 μg per close.

A formulation comprising QS2 I and liposomes may be prepared, for example containing a charged lipid, which increases the stability of the lipsomc-QS2 l structure for liposomes composed of saturated lipids. In these cases the amount of charged l ipid is oflen 1 -20% w/w. such as 5- 10%. The ratio of sterol Io phospholipid is 1 -50% (mol/mol), such as 20-25%. These compositions may contain MPL (3-deacylated mono-phosphoryl l ipid A. also known as 3D-MPL).

The saponins may be separate in the form of micelles, mixed micelles (generally, but not exclusively with bile salts) or may be in the form of ISCOM matrices (EP 0109942), liposomes or related colloidal structures such as worm-like or ring-like multimeric complexes or lipidic/layered structures and lamellae when formulated with cholesterol and lipid, or in the form of an oil in water emulsion (for example as in WO 95/17210). The saponins may often be associated with a metallic salt, such as aluminium hydroxide or aluminium phosphate (WO 98/ 15287).

Usually, the saponin is presented in the form of a l iposome, ISCOM or an oil in water emulsion.

Immunostimulatory oligonucleotides may also be used. Examples of oligonucleotides for use in adjuvants of the present disclosure include CpG containing oligonucleotides, generally containing two or more dinucleotide CpG motifs separated by at least three, more often at least six or more nucleotides. A CpG motif is a cylosine nucleotide followed by a guanine nucleotide. The CpG oligonucleotides are typically deoxynucleotides. In one embodiment the internucleotide in the oligonucleotide is phosphorodithioatc, or more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the disclosure. Also included within the scope of the disclosure arc oligonucleotides with mixed inlei nucleotide linkages. Methods for producing phosphorothioate oligonucleotides or phosphorodilhioate are described in US5.666. 153, US5,278,302 and WO 95/26204.

Examples of oligonucleotides are as follows: TCC ATG ACG TTC CTG ACG TT (CpG 1826)

TCT CCC AGC GTG CGC CAT (CpG 1758)

ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG

TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006)

TCC ATG ACG TTC CTG ATG CT (CpG 1668) TCG ACG TTT TCG GCG CGC GCC G (CpG 5456), the sequences may contain phosphorothioate modified internucleotide linkages.

Alternative CpG oligonucleotides may comprise one or more sequences above in that they have inconsequential deletions or additions thereto.

The CpG oligonucleotides may be synthesized by any method known in the art (for example see EP 468520). Conveniently, such oligonucleotides maybe synthesized utilising an automated synthesizer.

Examples of a TLR 2 agonist include pcptidoglycan or lipoprotein. Iinidazoquinolincs, such as Imiquimod and Rcsiquimod arc known TLR7 agonists. Single stranded RNA is also a known TLR agonist (TLR8 in humans and TLR7 in mice), whereas double stranded RNA and poly IC (polyinosinic-polycytidylic acid - a commercial synthetic mimetic of viral RNA) are exemplary of TLR 3 agonists. 3D-MPL is an example of a TLR4 agonist whilst CpG is an example of a TLR9 agonist.

An immunostimulant may alternatively or in addition be included. In one embodiment this immunostimulanl will be 3-deacylated monophosphoryl lipid A (3D-MPL).

Adjuvants combinations include 3D-MPL and QS2 I (EP 0 671 948 Bl), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/ 17210, WO 98/56414), or 3D-MPL formulated with other carriers (EP 0 689 454 Bl) including liposomes. Other adjuvant systems comprise a combination of 3D-MPL, QS2 I and a CpG oligonucleotide as described in US 6558670 and US 6544518.

In one aspect the adjuvant comprises 3D-MPL. In one aspect the adjuvant comprises QS2 1 . In one aspect the adjuvant comprises CpG. In one aspect the adjuvant comprises QS2 I and 3D-MPL. In one aspect the adjuvant comprises QS21 , 3D-MPL and CpG

In one aspect the adjuvant is formulated as an oil in water emulsion. In one aspect the adjuvant is formulated as liposomes.

The amount of 3D-MPL used is generally small, but depending on the vaccine formulation may be in the region of 1 to l OOOμg per dose, generally 1 to 500μg per dose, and more such as between 1 to lOOμg per dose ( 10, 20, 30, 40, 50, 60, 70, 80 or 90μg per dose).

The amount of CpG or immunoslimulatory oligonucleotides in the adjuvants or vaccines of the present disclosure is generally small, but depending on the vaccine formulation maybe in the region of 1 to lOOOμg per dose, generally 1 to 500μg per dose, and more such as between 1 to lOOμg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90μg per dose).

The amount of saponin for use in the adjuvants of the present disclosure may be in the region of 1 to l OOOμg per dose, generally 1 to 500μg per dose, more such as 1 to 250μg per dose, and more specifically between 1 to lOOμg per dose ( 10, 20, 30, 40, 50, 60, 70. 80 or 90μg per dose).

Thus in one embodiment there is provided a formulation comprising phosphoantigen and MPL.

In one embodiment there is provided a formulations comprising phosphoantigen and QS2 I .

In one embodiment there is provided a formulation comprising phosphoantigen and CpG.

Thus in one embodiment there is provided a formulation comprising phosphoantigen and MPL and QS21 .

Thus in one embodiment there is provided a formulation comprising phosphoantigen and MPL and CpG.

Thus in one embodiment there is provided a formulation comprising phosphoantigen and QS21 and CpG.

Thus in one embodiment there is provided a formulation comprising phosphoantigen and MPL, QS21 and CpG.

The above formulations may optionally comprise an antigcn/immunogcn provided that the antigen or immiinogcn is not specific to F. tiilarensis.

In one embodiment the phosphoantigen/formulation/composiiion is a vaccine.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Vollcr et al. University Park Press, Baltimore. Maryland. U.S.A.. 1978. Encapsulation within liposomes is described, for example, by Fiillcrton. US 4.235.877.

In one embodiment the formulation is provided as a formulation for topical administrations including inhalation. Suitable inhalablc preparations include inhalable powders, metering aerosols containing propel 1 ant gases or inhalablc solutions free from propcllant gases. Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the abovementioned active substances with physiologically acceptable excipient.

These inhalablc powders may include monosaccharides (e.g. glucose or arabinosc), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol, mannilol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another. Mono- or disaccharides are preferably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.

Particles for deposition in the lung require a particle size less than 10 microns, such as 1 -9 microns suitably from 0. 1 to 5 μm. particularly preferably from I to 5 μm. The particle size of the active (that is the antigen is ol primary importance).

The propellent gases which can be used to prepare the inhalable aerosols are known from the prior art. Suitable propellent gases arc selected from among hydrocarbons such as n-propane. n-butane or isobutanc and halohydrocarbons such as chlorinated and/or fluorinatcd derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutanc. The abovementioned propellent gases may be used on their own or in mixtures thereof.

Particularly suitable propellent gases arc halogcnatcd alkanc derivatives selected from among TG U . TG 12. TG 134a and TG227. Of the abovementioned halogcnatcd hydrocarbons. TG 134a ( 1 , 1.1 ,2-tctrafluoiOcthanc) and TG227 ( 1 , 1 , 1 ,2,3,3,3- hcptafluoropropanc) and mixtures thereof are preferred according to the invention.

The propellant-gas-containing inhalable aerosols may also contain other ingredients such as cosolvcnts, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients arc known in the art.

The propellant-gas-containing inhalable aerosols according lo the invention may contain up to 5 % by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5 % by weight. 0.01 to 3 % by weight, 0.01 5 to 2 % by weight, 0. 1 to 2 % by weight, 0.5 to 2 % by weight or 0.5 to I % by weight of active.

In one embodiment of the disclosure the phosphoantigen is employed in a prime boost regime, as the priming and/or boosting dose. Priming in this context refers to priming the immune system for a response, such as Vγ9Vδ2 T cel l proliferation and/activation. Boosting refer to increasing or sustaining the immune response of said priming.

Whilst not wishing to be bound by theory if the appropriate number of doses is exceeded in a relatively short period of time then it is possible induce anergy in the immune system, which is undesirable and should be avoided. In the one embodiment the dose is in the range lpg to lOOOμg per Kg, such as Ing to 10 μg per Kg.

In one embodiment the disclosure provides use of a phosphoantigcn for enhancing protective host immune responses specifically those mediated by Vγ9δ2 T cells to enable the host to fight infection with intracellular pathogens such as Francisella tulareiisis.

In one embodiment the disclosure provides a method of stimulating an immune response to fight Francisella ntlarensi.s infection.

In one embodiment the bioburden is lowered in vivo after administration of the phosphoantigen of the disclosure.

EXAMPLES

Materials and methods

Preparation of Peripheral Blood Mononuclear Cells (PBMCs)

Human blood (60ml) was collected into BD Vacutaincr CPT® sodium citrate lubes and centrifuged (25min: 1500g (no brake)). Buffy coats were removed, placed in Falcon tubes and centrifuged (300g: I5min). The cell pellet was resuspendcd in IOml medium (RPMI +10% Foetal calf scrum (FCS). 200U/ml penicillin.200μg/ml streptomycin and L-glutamine (1OmM)). Cell numbers were counted with trypan blue and centrifuged (300g: I5min).

Isolation ofyδ T cells from PBMC preparations γδ T cells were isolated from PBMC preparations using human Anli-TCRγ/δ Microbcad Kit (Millcnyi Biotech. UK) according to the method described below. The PBMC pellet was resuspendcd in 40μl medium and lOμl anti-TCR γδ Hapten-Antibody per IxIO⁷ cells, mixed well and refrigerated for lOmin. Medium (30 μl) and MACS anti-hapten microbeads-FITC (20 μl) per IxIO⁷ cells were added to cells and mixed well and refrigerated for I5min. Medium was added to cells and centrifuged (300g: 15min). The cell pellet was resuspendcd in 500 μl medium. Media (500 μl) was added to MACS column (MS 130-042-201: Miltcnyi Biotcc. UK). A BD Falcon cell strainer (70 μm) was placed on top of the column and the cell suspension added. The cell effluent was collected. The column was washed 3 times with medium (500 μl). The column was removed from the magnetic separator and place in a Bijoux. Medium (ImI) was added to the column and immediately flushed through using plunger from column kit. This is positive/enriched fraction contained the γδ T cells. The number of cells in positive fraction was counted.

Culture ofγδ T cells

The γδ T cell population was cultured in a 24-well plate in medium (RPMI + 10% Foetal calf serum (FCS).200U/ml penicillin. 200μg/ml streptomycin and L-glutamine (1OmM)) supplemented with 100U/ml human recombinant intcrleukin-2 (IL-2) (Sigma. UK) and isopcntcnyl pyrophosphonatc (IPP) 3μg/ml (Sigma. UK). Additional IL-2 (100 U/ml) was added to cells every 3 days. Cells were removed from wells by gently pipetting and scraping the bottom of the well with the tip to mix and break up cell clumps. Cells were counted and resuspended in ImI fresh medium without antibiotics to the correct cell concentration (IxIO⁶ cells/ml). THP-I human monocyte cell line

The non-adheient human monocytic cell line THP J (ATCC®) (ECACC UK) was giown in RPMI 10% Foetal calf sciurn (FCS) 200U/ml penicillin with oi without 200μg/ml sticptomycin and L glutamine (1OmM) all obtained fiom Sigma UK in cell cultuie flasks undei stenle conditions Pnoi to infection THP-I cells weie lemoved fiom cell cultuie flasks and centiifuged (30Og 15mm) Cells weie iesuspended in medium without antibiotics counted and readjusted to iequned cell concenttation (2x10⁶CcI Is/ml) THP-I cells (I ml/well) weie plated into 24 well plates and activated with PMA (phoibol 12-myiistatc 13-acclatc Sigma UK) at a final conccntiation of lOng/ml and weie incubated overnight (37C WoCOi) lo pioducc an adhcicnt monolayci Media was icmovcd fiom wells pi ioi to infection and ieplaced with fiesh media (900μl) without antibiotics

Giowth of F tulaiensis I VS

A viable frozen cultuie of / lulaiensis LVS was thawed and 40ul diops wcic spicad on dned BCGA plates at ACDP containment level 2 Plates weie incubated overnight (37"C WeCOi) Loops of bacteπal lawn weie taken and placed in PBS to make a cloudy suspension with an OD_430I11n leading of between 02-03 (l-2xlθ⁶ cfu/ml) A I IO dilution of the suspension was peifoimed pnoi to infection of THP-I to provide an appioximate infection dose of IxIO¹ cfu/ml

Gi owth of F iiilω en sis SCHU S4 A viable frozen cultuie ol / lulaiensis SCHU-S4 was thawed and 4OuI diops weie spicad on dned BCGA plates All woik with this oiganism was peifoimed at ACDP containment level 3 Loops of bacterial lawn weie taken and placed in PBS to make a cloudy suspension with an OD_41OnI,_! leading of between 02-03(12\IO⁶ cfu/ml) A I 10 dilution of the suspension was peifoimed pnoi to infection oi THP-I to piovidc an appioximaic infection dose of IxIO^{^} cfu/ml

Infection of IHP-I monoknei

F tulcnensis suspension (lOOμl /well) was added to the THP-I monolayei and incubated (30-45min 37⁰C COT) Media was then lemoved and fiesh media without antibiotics (iOOμl) was added Isolated γδ T cells oi media only weie added to the infected THP-I monolayei and incubated in a sealed containei (uith COi packet) in 37°C mcubatoi Ioi 24 h Supcinatanls wcic icmovcd fiom wells following incubation and sloicd at -20"C Ioi luithci baclciiological analysis Cells wcic lyscd using stenle distilled watei (ImI) pipetting up and down seveial times to disiupt cells The lysed cell suspension (lOOμl) was added to PBS (900μl) to pievent fuithei cell lysis Dioplets of the lysed cell suspension stabilised in PBS oi thawed cell cultuie supeinatants (3x20μl) weie placed onto duplicate BCGA plates and incubated foi S days (37°C WoCOo) Viable colonics wcic then counted

Results

Human γδ T cells and giowth of F tulaiensis

The effect of human γδ T cells tieated with IPP+1L-2 on intiacellulai giowth of F tukiiensis LVS was investigated In the absence ol γδ T cells the numbci of intiacellulai baclciia infecting the human monocytic cell line (THP-I) was 1000-10000 / lulaiensis LVS cfu/ml In the picscncc of puiificd human vδ T cells (2\ K)^"1 cells/ml) obtained fiom 6 dillcicnl individuals a significant dccicasc in the numbei of intiacellulai bacteiia was obseived (p<0000l) |ANOVA| in compaiison with media only contiols (Figuie IA) No significant diffeiences weie obseived between individuals in then ability to kill / tulaiensis LVS (p=0608) A icduciion in giowth of / lulaiensis SCHU S4 was also obseived in the piesence of human γδ T cells obtained fiom 3 individuals in comparison with media only controls (Figuie IB)

The number of bacteiia ieleased fiom THP-I duiing the infection assay was measured in supeinatants fiom wells containing puiified γδ T cells oi media alone In the absence of γδ T cells 1000- 10000 cfu F tulaiensis LVS /ml weie identified No bacterial giowth was obsei\ed in supeinatants letneved fiom wells containing γδ T cells liom S individuals

Figure 1 shows human γδ T cell mediated killing of / tulaiensis LVS and / tulaiensis SCHU S4 THP-I cells infected with F

(IxlC cfu/ml IxIO⁶ THP-I) were incubated foi 24 houis in the presence of γδ T cells (♦) oi media alone (α) A) Intiaccllulai / tulaiensis LVS lelπevcd following lysis of cells following infection Data is shown as mean of tiiplicate wells foi each individual (1-6) and any iepeats peifoimed on each individual aie also displayed B) Intiacellulai F tularensis SCHU S4 letneved following lysis of cells following infection fiom 3 difffeient individuals Data is icpiescnlcd as mean of tiiplicate wells for each individual (1-3) Enoi bais iepiesent SD of tiiplicate wells/individual

Figure 2 shows human γδ T cell mediated contiol of free F tularensis LVS Release of F tulaiensis LVS was investigated in supeinatants fiom THP I cells infected with F tulaiensis (IxIO¹ cfu/ml IxIO⁶THP 1) incubated foi 24houis Individual data showing mean of tiiplicate supeinatants (n=5) in the piesence of γδ T cells (♦) oi media alone (π) Enoi bais iepiesent SD of tiiplicate wells/individual

It is envisaged thai one oi moie embodiment desciibed heiein may be combined, as technically appiopπale In the context of this specification compiismg is to be intcipictcd as including

Aspects of the disclosuie coinpiising ceitain elements aie also intended to extend to alternative embodiments "consisting oi consisting essentially of the lelevant elements

Claims

1. A phosphoaniigen for ihe prophylaxis and/or treatment of tularemia.

2. A phosphoantigen according to claim 1, wherein the phosphoantigen is IPP.

3. A phosphoantigen according to claim 1, where in the phosphoantigen is BrHPP or IHPP.

4. A pharmaceutical composition comprising a phosphoaniigen as recited in any one of claims 1 to 3.

5. The pharmaceutical composition of claim 4, further comprising IL-2.

6. The pharmaceutical composition of either claim 4 or 5 for use in the prophylaxis and/or treatment of tularemia.

7. The pharmaceutical composition of claim 6 wherein the composition does not further comprise an antigen that is pharmaceutically active in the prophylaxis and/or treatment of tularemia.

8. A method of treatment for tularemia comprising administering a therapeutically effective amount of a phosphoantigen prophylactically or as treatment to a patient in need thereof.

9. A method of treatment for tularemia comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 4 to 7 prophylactically or as treatment to a patient in need thereof.

10. Use of the pharmaceutical composition of either claim 4 or 5 in the manufacture of a medicament for the prophylaxis or treatment of tularemia.