WO2005027958A1

WO2005027958A1 - Thymosin-beta-sulphoxide as an immunosuppressive active agent

Info

Publication number: WO2005027958A1
Application number: PCT/GB2004/003950
Authority: WO
Inventors: Peter John Dupont; Anthony Nigel Warrens; Mohantha Deevan Dooldeniya
Original assignee: Imperial College Innovations Ltd
Priority date: 2003-09-19
Filing date: 2004-09-15
Publication date: 2005-03-31

Abstract

The invention provides the use of thymosin beta-4 sulphoxide in the preparation of an immunosuppressant for prevention and/or treatment of lymphocyte-mediated organ or bone marrow transplant rejection. The invention also provides the use of thymosin beta-4 sulphoxide in the preparation of an immunosuppressant for the treatment of lymphocyte-mediated graft-versus-host disease.

Description

^■OTΗIOSΪN- BETA- SULPHOXIDE AS AET IMWIJISIDSUPPRKSølVE ACTIVE AGKHT

The present invention relates to therapeutic uses of thymosin beta-4 sulphoxide (Tβ SO) as an immunosuppressant. In particular, the invention relates to the use of thymosin beta-4 sulphoxide in the prevention and treatment of transplant rejection and graft-versus-host disease.

Transplantation of organs is used in the treatment of diseases such as cancer, end- stage renal failure, terminal cardiac failure, hepatic cirrhosis, burns, cystic fibrosis and leukaemia. Unless the donor and recipient are genetically identical, the graft antigens will elicit an immunological rejection response.

Bone marrow transplants pose an additional problem because they can result in graft- versus-host disease. Graft-versus-host disease is induced by immunologically competent T cells being transplanted into allogeneic recipients which are unable to reject them. This inability may be due to the genetic differences between the donor and recipient or because of a lack of immunocompetence of the recipient. The immunocompetent T cells transplanted with the bone marrow can attack the recipient. Graft-versus-host disease is a major complication of bone marrow transplantation causing severe damage, particularly to the skin and intestine.

Immunosuppressant drugs are administered to transplant patients to help prevent rejection of the graft or transplanted organ and in order to prevent the development of graft-versus-host disease.

The most widely used immunosuppressant drags are steroids, cyclosporin and azathioprine. Cyclosporin is a fungal macrolide produced by soil organisms which acts by selectively suppressing activated T lymphocytes, mainly by blocking cytokine production. Steroids act by suppressing macrophage function, restraining the clonal proliferation of Th cells and decreasing the transcription of many cytokine genes. Azathioprine is a pro-drug of 6-mercaptopurine and acts by inliibiting clonal proliferation in the induction phase of the immune response by a cytotoxic action on dividing cells.

The above drags however have unwanted side effects and since they impair immune responses, they also carry the hazard of a decreased response to infection and an increased risk of cancers. No current regime achieves complete prevention of rejection and most are associated with significant side-effects. There is therefore a need for alternative drugs and therapies to prevent transplant rejection.

In 1999, Young and colleagues (Nature Medicine Dec; 5:1424-7 (1999)) reported that they had purified a peptide factor with potent anti-inflammatory properties from supernatants of monocytes exposed to glucocorticoids and identified the peptide thymosin beta-4 sulphoxide (Tβ₄SO) as the active molecule. They went on to demonstrate that Tβ₄SO has potent anti-inflammatory effects both in vitro and in vivo through the inhibition of neutrophil chemotaxis. This was further reported in WO

99/49883 which relates to thymosin beta 4 sulphoxide as a pharmaceutical formulation and its use in treating inflammatory conditions.

It has now been found that thymosin beta-4 sulphoxide also has an immunosuppressant effect and can be used to protect transplanted tissues from rejection.

The immunomodulatory effects have been characterised in vivo in a murine model of skin allotransplantation and in vitro using a mixed leucocyte reaction. Mice treated systemically with thymosin beta-4 sulphoxide enjoyed prolonged allograft survival.

Intriguingly, pre-treating donor mice with T cells prolonged allograft survival even where the recipients remained untreated. Prolongation of graft survival was similar irrespective of whether it was the donor or recipient that received treatment.

In contrast to its anti- inflammatory actions which are mediated via its effect on neutrophils, the immunosuppressant effects of thymosin beta-4 sulphoxide are mediated via its effects on T lymphocytes and antigen-presenting cells (APCs) e.g. dendritic cells (DCs). As a result, thymosin beta-4 sulphoxide inhibits CD4⁺ T lymphocytes which are central to the process of graft rejection. Thymosin beta-4 sulphoxide is likely to exert its effects by interfering with recipient T cell priming by APCs. We have shown that Tβ SO inhibits DC migration in vitro. In vivo, it may inhibit trafficking of passenger APCs to recipient draining lymph nodes thereby interfering with the direct pathway of allorecognition.

Thymosin beta-4 sulphoxide may prove to be a useful addition to our immunosuppressive armamentarium with a novel tolerogenic effect that might complement the action of conventional immunosuppressants.

According to a first aspect of the invention, there is provided the use of thymosin beta-4 sulphoxide in the preparation of an immunosuppressant for prevention and/or treatment of lymphocyte-mediated transplant rejection.

The thymosin beta-4 sulphoxide may be provided as an oxidised form of thymosin beta-4, a biologically active fragment. Extra thymic occurrence of thymosin beta-4 has been reported by E Hannappel et al, Proc. Nat. Acad. Sci. USA 79, 2172 (1982). The amino acid sequence of thymosin beta-4 can be found in T.L.K. Low et al, Proc Natl Acad Sci USA 79, 1162 (1981). Automated solid phase synthesis of thymosin beta-4 is described in S.S. Wang et al Int. J Peptide Protein Res. 18, 413 (1981) and T.L.K. Low et al Biochemistry 22, 733(1983). In vitro synthesis of beta-4 by rat spleen mRNA is described in A.W. Filipowicz, B.L. Horecker Proc. Nat. Acad. Sci. USA 80, 1811 (1983). Biosynthesis of beta 4 by spleen cells is described in G.Xu et al Proc Natl Acad Sci USA 79, 4006 (1982).

The sequence of thymosin beta-4 is as follows:

SDJ^DMAEIEKFDKSKLKKTETQEI NPLPSKETIEQEKQAGES. Oxidation of a methionine residue of thymosin beta-4 located 6 amino acids from the N terminus converts the residue to methionine sulphoxide. The sequence of thymosin beta-4 sulphoxide is shown below. M represents the oxidised methionine residue.

SDKPDM^*AEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES

The methionine residue may however be further oxidised to form methionine sulphone which can act as an immunosuppressant in the same way as thymosin beta-4 sulphoxide. Further modifications of the methionine residue such as complexing the sulphur with metals may also result in an active agent for use as an immunosuppressant.

Physiologically active variants of the oxidised thymosin beta-4 sulphoxide are variants displaying the same or similar physiological properties as the thymosin beta- 4 sulphoxide. Preferably, such variants would include the oxidised methionine, but may be truncated, deleted or mutated forms thereof.

The thymosin beta-4 sulphoxide of the present application may therefore be formed through the reaction of native thymosin beta-4 under oxidising conditions. These oxidising conditions may comprise treatment with hydrogen peroxide. Preparation of thymosin beta-4 sulphoxide in this way may result in a mixture of non-oxidised thymosin beta-4 and thymosin beta-4 sulphoxide, however the preparation preferably comprises oxidised thymosin beta-4 only.

Thymosin beta-4 could be obtained from supematants of steroid-treated monocytes of mammalian origin e.g. bovine, equine, murine or human. Since thymosin beta-4 from these species are identical in sequence, thymosin beta-4 sulphoxide may be prepared from native thymosin beta-4 from one species and subsequently oxidised and formulated for administration to another species.

The thymosin beta-4 sulphoxide medicament will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient).

The thymosin beta-4 sulphoxide may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical

(including buccal, sublingual or transdermal), or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with a carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syraps or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions)

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For the preparation of solutions and syraps, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions oils

(e.g. vegetable oils) may be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986). Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. When formulated in an ointment, the active ingredient may be employed with either a paraffimc or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouthwashes. Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid including a coarse powder having a particle size for example in the range of 20 to 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 compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

Dosages of the substance of the present invention can vary between wide limits, depending upon the age and condition of the individual to be treated and a physician will ultimately determine appropriate dosages to be used.

This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

An effective dose for administration may comprise from 0.001 to 120mg/kg bodyweight, preferably 0.01 to 120 mg/kg and more preferably within the range of 0.01 to 50mg/kg, for example 0.05 to 20mg/kg.

The dosage may be administered to a transplant recipient during and/or following transplantation of the organ or bone marrow in order to prevent and/or treat transplant rejection or graft versus host disease. The dosage may also be administered to a transplant recipient prior to transplantation.

Alternatively, the dosage may be administered to the transplant donor prior to removal of the organ to be transplanted. In our experimental model, prolongation of graft survival was similar irrespective of whether it was the donor or recipient that received treatment.

The dosage may also be administered to both the transplant donor prior to removal of the organ to be transplanted and to the recipient following transplantation. In the context of this application, prevention of transplant rejection encompasses both the complete prevention of rejection, delay of rejection of the transplant or reduction in the severity of rejection episodes.

Treatment of transplant rejection encompasses the treatment of the recipient following onset of symptoms of rejection.

The thymosin beta-4 sulphoxide is suitable for use in the treatment of rejection of any transplantable tissue or organ. These tissues or organs include the kidney, heart, lung, heart/lung, liver, cornea, pancreas, islets of Langerhans, bone marrow, small bowel and skin.

In a second aspect of the invention there is provided the use of thymosin beta-4 sulphoxide as an immunosuppressant.

The thymosin beta-4 sulphoxide of the first and second aspects preferably acts by inhibiting the proliferation of CD4⁺ T cells.

In a third aspect of the present invention there is provided the use of thymosin beta-4 sulphoxide in the preparation of an immunosuppressant for the treatment of lymphocyte-mediated graft-versus-host disease.

In a fourth aspect of the present invention there is provided a method for the prevention and/or treatment of lymphocyte-mediated transplant rejection comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a transplant recipient. By transplant recipient it is meant a person into whom an organ is transplanted following its removal from a donor.

In a fifth aspect of the present invention there is provided a method for the prevention and/or treatment of lymphocyte-mediated graft-versus-host disease comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a bone marrow transplant recipient. By bone marrow recipient it is meant a person into whom bone marrow is transplanted following its removal from a donor.

In a sixth aspect of the present invention there is provided a method for the prevention and/or treatment of lymphocyte-mediated transplant rejection comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a transplant donor prior to removal of the tissue or organ to be transplanted.

In a final aspect of the present invention there is provided a method for the prevention and/or treatment of lymphocyte-mediated graft-versus-host disease comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a transplant donor prior to harvesting of the bone marrow.

In a particularly preferred embodiment of the invention there is provided an improved method of preparing an organ or preparing bone marrow for transplant comprising administering a therapeutic amount of thymosin beta-4 sulphoxide to an organ or bone marrow donor and then removing the organ or harvesting the bone marrow for transplant to a recipient.

In a further particularly preferred embodiment of the invention there is provided an improved method of transplanting an organ or transplanting bone marrow comprising transplanting the donor tissue into the transplant recipient and then administering a therapeutic amount of thymosin beta-4 sulphoxide to the transplant recipient.

In a further particularly preferred embodiment of the invention there is provided an improved method of transplanting an organ or transplanting bone marrow comprising administering a therapeutic amount of thymosin beta-4 sulphoxide to an organ or bone marrow donor, removing the organ or harvesting the bone marrow, transplanting the donor tissue into the transplant recipient and then administering a therapeutic amount of thymosin beta-4 sulphoxide to the transplant recipient. The above improved methods result in treatment or prevention of lymphocyte- mediated transplant rejection or lymphocyte-mediated graft-versus-host disease.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be constraed as being limiting on the invention. Reference is made to a number of Figures in which:

FIGURES la, lb and lc shows the effect of treatment of recipient mice with thymosin beta-4 sulphoxide on the inhibition of rejection of mouse skin allografts across a major MHC mismatch, a multiple minor mismatch or single minor mismatch.

FIGURE 2 shows the effect of pre-treatment of donor mice with thymosin beta-4 sulphoxide prior to removal of the skin on the inhibition of rejection of mouse skin allografts across a multiple minor mismatch.

FIGURE 3a shows the proliferation of CD4+ T cells derived from mice treated with thymosin beta-4 sulphoxide in comparison to those of PBS-treated controls.

FIGURE 3b shows the results of recipient CD4+ cells isolated at various time points and used as responders in an MLR with either donor or third party dendritic cells as stimulators.

FIGURE 4 shows the proliferation of T cells from untreated recipient mice that had received grafts from thymosin beta-4 sulphoxide treated donors.

FIGURE 5a shows the effect of thymosin beta-4 sulphoxide on the hihibition of human T cell proliferation FIGURE 5b shows the purity of isolates of human T cells

FIGURE 5c shows the effect of thymosin beta-4 sulphoxide on the activation of human T cells.

FIGURE 6 shows the inhibition of dendritic cell migration by Thymosin beta-4 sulphoxide.

Example 1: The effect of thymosin beta-4 sulphoxide treatment of recipient mice in inhibiting mouse skin allograft rejection.

Mouse skin allografting

Skin allotransplants were performed according to the Medawar method. Skin was transplanted from one strain of mouse onto individuals of a different strain Donor tail skin was removed and placed onto the graft bed prepared on the recipient. The mice were split into active treatment and placebo groups. Mice in the active treatment group received intraperitoneal injections of Tβ₄SO (0.5-2μg) at the time of transplantation and then every 48 hours up to a maximum of 10 doses (day 18). The placebo group of mice received placebo injections of the carrier solution (phosphate buffered saline) on the same timescale. The grafts were covered with a plaster of

Paris cast which was removed on day 6-10 and the grafts were subsequently checked daily until all were rejected.

The skin allotransplants were repeated, varying the degree of difference in tissue type between donor and recipient mice. Grafts were either transplanted between very different mouse strains ("major MHC mismatch" e.g. C57/B16 to BALB/c) which elicits a vigorous rejection response or between very similar strains which induces a less vigorous rejection response ("minor mismatch" e.g. C57/B16 male to C57/B16 female; or "multiple minor mismatch" e.g. C57/B16 to BALB/b ). Figure la shows the results of skin allotransplants performed across a major MHC mismatch (C57BL/6 donor to BALB/c recipient). The mean graft survival with Tβ₄SO was 15.9 days, compared with placebo at 11.5 days (p=0.005 Log rank test).

Figure lb shows the results of skin allotransplants performed across a multiple minor mismatch (C57/B16 donor to BALB/b recipient). The mean graft survival with Tβ₄SO was 20.3 days compared with 14.7 days for placebo treated mice (p=0.03 Log rank test)

Figure lc shows the results of skin allotransplants performed across a single minor mismatch (C57/B16 male to C57/B16 female). The mean graft survival with Tβ SO was 51 days compared with 39 days for placebo treated mice (p=0.002 Log rank test)

Mice receiving active treatment with Tβ SO enjoyed a statistically significant prolongation of skin graft survival in each type of transplant (p<0.05 in all cases; Log

Rank Test). When the immunological disparity between donor and recipient was reduced by transplanting across a single or multiple minor histocompatibility barriers rather than an MHC difference, the quantitative difference in graft survival between Tβ SO-treated and control groups was more pronounced . Treatment with thymosin beta-4 sulphoxide protects grafts from rejection i.e. it suppresses the immune response to the graft.

Example 2: The effect of thymosin beta-4 sulphoxide treatment of donor mice in inhibiting mouse skin allograft rejection.

The skin allotransplants of Example 1 were repeated however, the donor mice were pre-treated with Tβ SO prior to removal of the tail skin for transplantation and the recipient mice were not treated. The donor mice were split into active treatment and placebo groups. Donor mice in the active treatment group received a course of 10 doses of intraperitoneal injections of Tβ₄SO (5μg) at 48 hours prior to skin graft donation. The placebo group of donor mice received placebo injections of the carrier solution (phosphate buffered saline) on the same timescale.

The skin allotransplants were again performed according to the Medawar method following treatment of the donor mice. Skin was transplanted across a multiple minor histocompatibility barrier and the recipient mice were left untreated. Donor tail skin was removed and placed onto the graft bed prepared on the recipient. The grafts were covered with a plaster of Paris cast which was removed on day 6-10 and the grafts were subsequently checked daily until all were rejected.

Figure 2 shows the results of the treatment of the donor mice prior to removal of the skin for transplantation. Graft survival was prolonged in the mice that received grafts from Tβ₄SO treated donors (p=0.001; Log rank test) and was comparable to graft survival in mice that received grafts from untreated donors but subsequently received Tβ₄SO treatment post transplantation. Treatment with thymosin beta-4 sulphoxide therefore renders the skin graft less immunogenic on transplantation.

Tβ₄SO prolongs graft survival irrespective of whether it is the donor or recipient mice that receives treatment.

Example 3: Mixed leucocyte reaction test: treatment of recipient mice

Purification of murine CD4⁺ lymphocytes

Using aseptic technique, the spleen and lymph nodes were removed from sacrificed mice and placed in RPMI 1640 medium (Gibco) with 10% foetal calf serum (MB Meldrum Ltd. UK). The tissues were macerated using a 70μm nylon cell-strainer mesh (BD Falcon) and the solution centrifuged for 10 minutes at 4°C at 1800rpm with brakes on. The resulting pellet was incubated with 1ml red blood cell lysis solution (Pure gene) at room temperature for 15 minutes to eliminate contaminating red blood cells.

Purification of CD4+ T cells was achieved using a negative depletion technique. Briefly, the cells were washed, resuspended in RPMI and 0.5 ml each of anti-CD8 antibody (53.6.72 hybridoma supernatant) and anti-MHC class II antibody (M5/114.15.2 hybridoma supernatant) added. The cells were incubated with the antibodies for 30 minutes at 4°C. Following incubation, the cells were washed with RPMI and the pellet resuspended in 1ml of goat anti-rat IgG .coated magnetic microbeads (Bio ag) at a concentration of 20 beads/cell, Again the cells were incubated at 4°C for 30 minutes then placed in a magnet and the supernatant containing CD4+ T cells removed. A further round of negative depletion produced a final CD4+ T cell purity of > 90%.

Mixed leucocyte reaction (MLR)

A mixed leucocyte reaction was used to assess T cells functionally at a site distant to the transplant.

Dendritic cells (DC) were isolated from the bone marrow of (untreated) C57 B16 mice and incubated for 5 days in RPMI. Lipopolysaccharide Ing/ml was added on day 5 to promote maturation. The DCs were washed and then irradiated with 35Gy. These cells were used as stimulators.

C57BL/6 tail skin was grafted onto BALB/b recipients (multiple minor mismatch). Recipient mice received intraperitoneal injections of either 5μg Tβ₄SO or PBS every

48 hours up to a maximum of 10 doses. These BALB/b mice were then sacrificed at various time points (day 5, 10, 20 or 30) and their spleens and lymph nodes removed. CD4+ T cells were then isolated from the murine spleen and lymph nodes as described above and used as responders in a MLR using DC from untreated C57BL/6 mice as stimulators. Cells were plated out into 96-well round-bottomed plates suspended in RPMJ/10% FCS with each well having a final volume of 250 l. The plates were then incubated for 4 days at 37°C with 5% CO₂. On day 5, the cells were pulsed with lμCi of tritiated thymidine. After 16 hours the cells were harvested and their proliferation assessed by tritiated thymidine incorporation as measured by liquid scintillation spectrometry.

The immune response of mice that received thymosin beta-4 sulphoxide treatment is suppressed compared with those who received placebo. Figure 3a represents proliferation of CD4+ T cells. T cells derived from Tβ SO-treated mice showed significantly less proliferation than those from PBS-treated controls.

Figure 3b shows the results of recipient CD4+ cells isolated at various time points and used as responders in an MLR with either C57BL/6 (donor) or CBA (third party) dendritic cells as stimulators. The data is presented as counts per minute (cpm) relative to the mean cpm in the control MLR (PBS) expressed as a percentage to facilitate comparison between the responses to donor and 3rd party.

The inhibition of proliferation shown in Figure 3a was not seen in response to the irrelevant third party stimulator suggesting that the mice had developed donor-specific hyporesponsiveness i.e. tolerance.

Example 4: Mixed leucocyte reaction test: pre- treatment of donor mice

The Mixed Leucocyte reaction test of Example 3 was repeated using T cells derived from untreated mice that had received grafts from Tβ₄SO-treated donors.

C57BL/6 donor mice received 10 doses of intrapeπtoneal injections of Tβ SO at 48- hour intervals. Tail skin was then removed and drafted onto untreated BALB/b mice. The recipient mice were then sacrificed at various time points following grafting and used as responders in an MLR with either C57BL/6 (donor) or CBA (third party) dendritic cells as stimulators (see Example 3).

Cells were plated out into 96-well round-bottomed plates suspended in RPMI/10%

FCS with each well having a final volume of 250μl. The plates were then incubated for 4 days at 37°C with 5% CO₂. On day 5, the cells were pulsed with IμCi of tritiated thymidine. After 16 hours the cells were harvested and their proliferation assessed by tritiated thymidine incorporation as measured by liquid scintillation spectrometry.

Figure 4 shows the proliferation of T cells. T cells derived from mice which received transplants from pre-treated donor mice showed significantly less proliferation than those from PBS-treated controls. Data is presented as cpm relative to the mean cpm in the control MLR (PBS) expressed as a percentage to facilitate comparison between the responses to donor and third party.

The immune response of mice that received transplants from thymosin beta-4 sulphoxide pre-treated donors is therefore suppressed compared with those who received placebo. Again the hyporesponsiveness is donor-specific and may reflect impaired T cell priming.

Example 5: T cell proliferation and activation assays Human T lymphocyte isolation

Blood was drawn from a healthy volunteer and peripheral blood mononuclear cells isolated by a standard Ficoll density gradient separation technique (Lymphoprep - Nycomed Ltd, UK). Fresh unactivated T cells were isolated by a negative depletion technique using Dynabeads according to manufacturer's instructions (Dynal Biotech, UK). T cells were stained with a fluorescein isothiocyanate (FITC) labelled mouse anti-human CD3 antibody or matched mouse IgGlK isotype control (both Sigma, UK) and purity assessed by flow cytometry using a FACScalibur instrument and Cell Quest software (both Becton Dickinson, UK), Isolates were consistently 90-95% pure (see Figure 5b).

Hisman T cell proliferation assay

10⁵ T cells were cultured together with an irradiated B cell line (WS5 - a kind gift from Dr G Lombard!) as stimulators. On day 5, the cells were pulsed with IμCi of tritiated thymidine then harvested 18 hours later and proliferation assessed by tritiated thymidine incorporation as measured by liquid scintillation spectrometry. Responders were taken from a normal volunteer. Tβ₄SO was added at the beginning of the culture at a dose of 150ng/ml.

Results are shown in Figure 5a. Tβ₄SO does not have any inhibitory effect on murine or human T cell proliferation in an MLR using a highly purified population of lymphocytes as responders (Figure illustrates human data).

Human T cell activation assay

T cells were isolated as described above. 5 x 10⁵ cells suspended in 5θ0 L of RPMI/ 10% FCS were placed in the wells of 24 well plate. Tβ₄SO (l-150ng/mL) or carrier (PBS) was added to the cultures and then the cells were stimulated using PMA

(5ng/mL) plus ionomycin lng mL. Upregulation of the T lymphocyte cell surface antigens CD69 and CD25 as early and late markers of T cell activation were assessed by flow cytometry following staining with FITC-conjugated mouse anti-human CD69 and CD25 antibodies (both Becton Dickinson, UK).

Figure 5c shows the effect of Tβ SO on T cell activation. Flow cytometric analysis of human T cells stimulated with PMA/ionomycin and stained for CD69 and CD25 showed no difference in the degree of upregulation of these early and late markers of T cell activation compared to the control. Example 6: Dendritic cell migration assay Human dendritic cell culture

Human PBMCs were isolated from buffy coats or from fresh blood by standard ficoll density gradient centrifugation technique and the monocytes/macrophages enriched by a plastic adherence step. The adherent population was cultured in RPME supplemented with 10% FCS (Sera Laboratories International, UK) plus supplemental L-glutamine 200mM (Invitrogen, UK) and antibiotics (penicillin 10,00Ou/rnL and streptomycin lOmg mL) (Invitrogen, UK) in the presence of re.combinant .GM-CSF (20ng mL) (Glaxo Smith Kline, UK) and IL-4 (10ng/mL) (First Link, UK).

Maturation was induced by overnight incubation with lipopolysaccharide (LPS) lOOng mL (Sigma, UK).

Dendritic cell migration assay For the migration assay, DCs were cultured as described above and pulsed with

Tβ₄SO 10-lOOng/mL or carrier (PBS) on days 0, 2 and 4, then matured by overnight incubation with LPS lOOng/mL. The cells were then harvested, washed and resuspended in ϊscove's modified Dulbecco's medium (Gibco) supplemented with 5% HS. 10⁵ cells were placed in the upper chamber of a transWell (8μm pore size) (Becton Dickenson, UK). The chemoattractant acrophage inflammatory protein lα (MTP-lα) (Peprotech Ltd, UK) suspended in Iscove's modified Dulbecco's medium 15% HS at a concentration of lOng/mL was placed in the lower chamber. The plates were incubated overnight and the cells appearing in the lower chamber were sampled and counted using a haemocytometer. Migration in the absence of chemokine was also assessed (medium alone in the lower chamber).

The results are shown in Figure 6. Tβ SO inhibited both spontaneous DC migration and DC migration in response to the chemoattractant MDP-la. Thymosin beta-4 sulphoxide therefore exerts an effect on dendritic cell function, which is likely to result in impaired T cell priming.

Claims

1. Use of thymosin beta-4 sulphoxide in the preparation of an immunosuppressant for prevention and/or treatment of lymphocyte- mediated transplant rejection.

2. Use of thymosin beta-4 sulphoxide as an immunosuppressant.

3. Use of thymosin beta-4 sulphoxide in the preparation of an immunosuppressant for the treatment of lymphocyte-mediated graft-versus- host disease.

4. Use of claims 1 to 3 wherein thymosin beta-4 sulphoxide inhibits the proliferation of CD4⁺ T cells.

5. Use of claims 1 or 4 wherein the transplant rejection is rejection of a transplanted organ or tissue selected from the group consisting of kidney, heart, lung, heart/lung, liver, cornea, pancreas, islets of Langerhans, bone marrow, small bowel and skin.

6. A method for the prevention and/or treatment of lymphocyte-mediated transplant rejection comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a transplant recipient.

7. The method of claim 6 wherein the transplant recipient receives an organ or tissue selected from the group consisting of kidney, heart, lung, heart/lung, liver, cornea, pancreas, islets of Langerhans, bone marrow, small bowel and skin.

8. A method for the prevention and/or treatment of lymphocyte-mediated graft- versus-host disease comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a bone marrow transplant recipient.

9. A method for the prevention and/or treatment of lymphocyte- mediated transplant rejection comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a transplant donor prior to removal of the tissue or organ to be transplanted

10. The method of claim 9 wherein the tissue or organ is selected from the group consisting of kidney, heart, lung, heart/lung, liver, cornea, pancreas, islets of Langerhans, bone marrow, small bowel and skin.

11. A method for the prevention and/or treatment of lymphocyte-mediated graft- versus-host disease comprising the administration of a therapeutic amount of thymosin beta-4 sulphoxide to a bone marrow transplant donor prior to the harvesting of the bone marrow.