AU2003261499B2 - Ex vivo treatment of allogeneic and xenogeneic T-cells with gp39 antagonists - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANTS: Regents of the University of Minnesota AND Trustees of Dartmouth College ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000 INVENTION TITLE: "Ex vivo treatment of allogeneic and xenogeneic T-cells with gp39 antagonists" The following statement is a full description of this invention, including the best method of performing it known to us: EX VIVO TREATMENT OF ALLOGENEIC AND XENOGENEIC T-CELLS WITH gp39 ANTAGONISTS Field of the Invention Methods of treating transplanted tissue or organs (allogeneic or xenogeneic) ex vivo in order to tolerize T-cell contained therein to donor antigens (xenoantigens or alloantigens) are provided. The treated tissue or organ can be transplanted in a recipient with reduced risk of graft-versus-host disease.

Background of The Invention To induce antigen-specific T-cell activation and clonal expansion, two signals provided by antigen-presenting cells (APCs) must be delivered to the surface of resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp.

Med. 165, 302-319; Mueller, et al. (1990)J. Immunol. 144,3701-3709; Williams, I.R. and Unanue, E.R. (1990) J. Immunol.145, 85-93). The first signal, which confers specificity to the immune response, is mediated via the T-cell receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, termed co-stimulation, induces T cells to proliferate and become functional (Schwartz, R.H. (1990) Science 248, 1349-1356). Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins, et al. (1988) J. Immunol. 140, 3324-3330; Linsley, et al.

(1991) J. Exp. Med. 173, 721-730; Gimmi, et al., (1991) Proc. Natl.

Acad. Sci. USA, 88, 6575-6579; Young, et al. (1992) J. Clin. Invest. 229-237; Koulova, et al. (1991) J. Exp. Med. 173, 759-762; Reiser, et al. (1992) Proc. Natl. Acad. Sci. USA, 89. 271-275; van-Seventer, et al.

(1990) J. Immunol. 144, 4579-4586; LaSalle, et al., (1991) J. Immunol.

147, 774-80; Dustin, et al., (1989) JExp. Med. 169, 503; Armitage, R.J., et al. (1992) Nature 357, 80-82; Liu, et al. (1992) J. Exp. Med. 175, 437- 445). One co-stimulatory pathway involved in T cell activation involves the molecule CD28 on the surface ofT-cells. This molecule can receive a costimulatory signal delivered by a ligand on B-cells or other APCs. Ligands for CD28 include members of the B7 family of B lymphocyte activation antigens such as B7-1 and/or B7-2 (Freedman, A.S. et al. (1987) J Immunol.

137, 3260-3267; Freeman, G.J. et al. (1989) J. Immunol. 143, 2714-2722, Freeman, G.J. et al. (1991)J. Exp. Med. 174, 625-631; Freeman, G.J. et al.

(1993) Science 262, 909-911; Azuma, M. et al. (1993) Nature 366, 76-79; Freeman, G.J. et al. (1993) J. Exp. Med. 178, 2185-2192). B7-1 and B7-2 are also ligands for another molecule. CTLA4, present on the surface of activated T cells, although the role of CTLA4 in co-stimulation is unclear.

Delivery to a T cell of an antigen-specific signal with a co-stimulatory signal leads to T-cell activation, which can include both T-cell proliferation and cytokine secretion. In contrast, delivery to a T-cell of an antigen-specific signal in the absence of a co-stimulatory signal is thought to induce a state of unresponsiveness or anergy in the T-cell, thereby inducing antigen-specific tolerance in the T-cell.

Interactions between T-cells and B-cells play a central role in immune responses. Induction of humoral immunity to thymus-dependent antigens requires "help" provided by T helper (hereafter Th) cells. While some help provided to B lymphocytes is mediated by soluble molecules released by Th cells (for instance lymphokines such as IL-4 and IL-5), activation of B cells also requires a contact-dependent interaction between B cells and Th cells.

Hirohata et al., J. Immunol., 140:3736-3744 (1988); Bartlett et al., J.

Immunol., 143:1745-1754 (1989). This indicates that B-cell activation involves an obligatory interaction between cell surface molecules on B-cells and Th cells. The molecule(s) on the T-cell therefore mediates contactdependent helper effector functions of the T-cell. A contact-dependent interaction between molecules on B-cells and T-cells is further supported by the observation that isolated plasma membranes of activated T-cells can provide helper functions necessary for B-cell activation. Brian, Proc. Natl.

Acad. Sci. USA, 85:564-568 (1988); Hodgkin et al., J. Immunol., 145:2025- 2034 (1990); Noelle et al., J. Immunol., 146:1118-1124 (1991).

A molecule, CD40, has been identified on the surface of immature and mature B lymphocytes which, when crosslinked by antibodies, induces B-cell proliferation. Valle et al., Eur J. Immunol., 19:1463-1467 (1989); Gordon et al., J. Immunol., 140:1425-1430 (1988); Gruber et al., J. Immunol., 142: 4144- 4152 (1989). CD40 has been molecularly cloned and characterized.

Stamenkovic et al., EMBO 8:1403-1410 (1989). A ligand for CD40, gp39 (also called CD40 ligand or CD40L and recently CD154) has also been molecularly cloned and characterized. Armitage et al., Nature, 357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101 (1992); Hollenbaugh et al., EMBO 11:4313-4319 (1992). The gp39 protein is expressed on activated, but not resting, CD4' Th cells. Spriggs et al., J. Exp. Med.

176:1543-1550 (1992); Lane et al., Eur. J. Immunol., 22:2573-2578 (1992); Roy et al., J. Immunol., 151:1-14 (1993). Cells transfected with the gp39 gene and expressing the gp39 protein on their surface can trigger B-cell proliferation and, together with other stimulatory signals, can induce antibody production. Armitage et al., Nature, 357:80-82 (1992); Hollenbaugh et al., EMBO 11:4313-4319 (1992).

Brief Description of the Invention Graft Versus Host Disease (GVHD) is a multi-organ system destructive process caused by the infusion of donor allogeneic T-cells into recipients. Because acute graft versus host disease occurs in 20-40% of recipients of HLA-identical sibling donor grafts and up to 70-80% of recipients of unrelated donor grafts, approaches to prevent this complication of bone marrow transplantation are needed. Two general type of strategies have been used to date. The first involves the in vivo infusion of immune suppressive agents such as methatrycide, cyclosporine A, and steroids. The acute graft versus host disease instances above are those observed during the infusion of these in vivo immune suppressive agents. In addition to their incomplete protective effects, these immune suppressive agents lead to prolonged periods of immune deficiency after bone marrow transplantation thereby re-exposing the recipient to infectious complications and potentially increasing the incidence of relapse after bone marrow transplantation. A second general approach has involved the ex vivo removal of T-cells from the donor graft. This approach while reasonably effective in preventing acute graft versus host disease results in a higher incidence of graft failure, relapse, infectious complications, and delays immune reconstitution time.

By contrast, the present invention is directed to a method of treating donor T-cells ex vivo, to render such T-cells substantially non-responsive to allogeneic or xenogeneic antigens upon transplantation into a host. More specifically, the present invention is directed to a method for treating donor Tcells ex vivo with an amount of at least one gp39 (CD 154) antagonist and allogeneic or xenogeneic cells or tissues, in order to render such T-cells substantially non-responsive to donor antigens (alloantigens or xenoantigens) upon transplantation into a host containing such allogeneic or xenogeneic cells.

The present invention thus provides an effective means of preventing or inhibiting graft-versus-host disease responses that would otherwise potentially occur upon transplantation of donor T-cells, or tissues or organs containing, donor bone marrow or peripheral blood cells into a recipient.

Preferably, donor T-cells will be incubated ex vivo with a sufficient amount of an anti-gp39 antibody and cells from the transplant recipient, for a sufficient time, to render the donor T-cells substantially non-responsive to recipient cells upon transplantation.

This will generally be accomplished by conducting a mixed lymphocyte reaction in vitro using donor T-cells and irradiated T-cell depleted host alloantigen or xenoantigen-bearing stimulators. To this culture will be added a gp39 antagonist, preferably an antibody or antibody fragment that specifically binds gp39 (CD154). Alternatively, the gp39 antagonist may comprise a soluble CD40 or soluble CD40 fusion protein, Brief Description of the Drawings Figure 1 shows the effect of anti-CD40L mAb treatment of donor Tcells in a primary MLR culture.

Figure 2A shows the effect of the addition of anti-CD40L (gp39) mAb on IL-2 production in primary MLR culture.

Figure 2B shows the effect of anti-CD40L mAb to gamma interferon production in a primary MLR culture.

Figure 3A shows the induction of anti-host alloantigen hyporesponsiveness by anti-CD40L mAb in secondary cultures is reversible by erogenous IL-2.

Figure 3B shows that donor T-cells exposed to anti-CD40 mAb in primary MLR culture have intact IL-2 responses in secondary culture.

Figure 4A shows the addition of anti-CD40L mAb to a primary MLR culture inhibits IL-1 production as measured in a secondary MLR culture.

Figure 4B shows that the addition of anti-CD40L mAb to a primary MLR culture inhibits gamma interferon production as measured in a secondary MLR culture.

Figure 5A shows that anti-CD40L mAb treatment of donor T-cells in an MLR culture markedly reduced in vivo GVHD capacity.

Figure 5B shows the effect of anti-CD40L treatment on mean body weight after transplantation.

Detailed Description of the Invention The following terms will be understood to have the following definitions: -7- Allogeneic Cell refers to a cell obtained from a different individual of the same species as the recipient.

Alloantigen refers to a cell obtained from a different individual of the same species as the recipient.

Xenogeneic cell refers to a cell obtained from a different species relative to another species, typically a transplant recipient. (For example, baboon T-cells would comprise xenogeneic cells if transplanted in a human recipient.) Xenoantigen refers to an antigen expressed by a cell obtained from a different species relative to another species, typically a transplant recipient.

gp39 antagonist refers to a molecule that interferes with the gp39 (CD154) CD40 interaction. A gp39 antagonist preferably will be an antibody directed against gp39 a monoclonal antibody specific to human gp39), or a fragment or derivative thereof Fab, F(ab)' 2 fragment, chimeric antibody, human antibody or humanized antibody). Also, gp39 antagonists include soluble forms of a fusion protein of a gp39 ligand soluble CD40Ig) or pharmaceutical agents that interfere with gp39 interaction.

g22 or CD 154 or CD40L or CD40CR is a molecule expressed on the surface of a T-cell that interacts with a molecule, CD40, identified on the surface of immature B-cell and mature B-cell lymphocytes that is involved in inducing B-cell proliferation. Specifically, the interaction with gp39 on Tcells with CD40 on B-cells plays a central role in activating B-cell responses to an antigen. Also, it has been discovered that gp39 plays a significant role in the response of T-cells to antigens, allo- and xenoantigens.

P:\OPERPrxk/003261499 claims 50 doc-21/12/05 -8-

O

T-Cell non-responsiveness or T-cell tolerance in the present invention refers to the reduced immune response (graft-versus-host response) elicited by donor T-cells against allo- or xenoantigen bearing cells upon transplantation of these donor T-cells into a recipient after they have been contacted ex vivo with a gp39 antagonist (anti-gp39 antibody) and xeno- or alloantigen bearing cells.

I As discussed, the present invention provides an alternative approach to the C prevention of graft-versus-host disease upon transplantation of foreign donor T-cell 0 containing compositions, allogeneic or xenogeneic bone marrow or peripheral blood cells.

It is known that a very small proportion of donor T-cells possess the capability to recognize host alloantigen. The present invention seeks to eliminate this response (render such cells non-responsive or tolerized to alloantigen or xenoantigen) by functionally altering the population of T-cells with allo- or xenoantigen reactive capabilities.

It is hypothesized by the present inventors that this could potentially be accomplished by initiating a mixed lymphocyte reaction of donor T-cells and irradiated T-cell depleted host alloantigen or xenoantigen bearing stimulators, and adding to this mixed lymphocyte reaction culture a gp39 antagonist, in particular an anti-gp39 antibody. The hope was that this would interfere with gp39 interactions in vitro (between donor T-cell and alloantigen or xenoantigen bearing cells), and render such cells tolerized or non-responsive to alloantigen or xenoantigen bearing cells upon transplantation into a transplant recipient. It was theorized that this would potentially be possible based on previous successful reports in the literature, including those of the present inventors, relating to inducing T-cell tolerance to allogeneic or xenogeneic tissue in vivo by the treatment of the transplantation recipient with gp39 antagonist (anti-gp39 antibody), alone or in combination with allogeneic or xenogeneic cells, prior, contemporaneous or subsequent to transplantation of xenogeneic or allogeneic tissue or organ.

This in vivo approach has been demonstrated to be highly effective for indicating T-cell tolerance to various tissues or organs, bladder, skin, cardiac tissue, et seq.

However, it was unpredictable whether this methodology could be extended to the induction of T-cell tolerance or non-responsiveness in vitro.

This outcome was not reasonably predictable because previous studies reported in the literature have demonstrated that there is no requirement that gp39 be present for the induction of in vitro T-cell activation. For example, Flavell and colleagues (Nature, 378:617-620 (1995)) have shown that T-cell receptor transgenic T-cells that were generically deficient in gp39 expression responded normally to antigen-presenting cells and antigen. This demonstrated that T-cell activation does not require gp39, and indeed can occur normally in the absence of gp39 in vitro.

S Specifically, data reported by Grewal, J.S. et al, Nature, 378:617-620 (1995), "Impairment of antigen-specific T-cell priming in mice lacking ligand" demonstrated that there is no requirement that gp39 be present for short term in vitro activation ofT-cells, and that allospecific cell T tolerance :can be generated in vitro in the absence of gp39.

Therefore, quite unexpectedly it has been shown that T-cell tolerance ;or non-responsiveness of donor T-cells can be effectively induced in vitro by P OP \PTA2 326I499 climrs 30.dc.2 I/I 2/ olo O incubating such cells with a gp39 antagonist and recipient allogeneic or xenogeneic cells which are depleted of recipient T cells. This technique affords tremendous potential in the treatment of transplant recipients since it affords a highly efficient, non-invasive means of rendering transplanted T-cells contained in transplanted tissue or organ tolerized or non-responsive to recipient alloantigens or xenoantigens.

Consequently, this transplanted tissue or organ, zenogeneic or allogeneic bone marrow should not elicit an adverse grant-versus-host response upon transplantation.

I Moreover, the fact that tolerance is induced in vitro is further advantageous as this treatment may be utilized in conjunction with other anti-rejection strategies, i.e.

cyclosporine or other immunosuppressants. Also, it may be combined with anti-gp39 antibody administration (or other ligand) prior, concurrent or subsequent to transplantation.

In fact, the subject method may eliminate the need for other anti-rejection drugs, which given their immunosuppressant activity, may result in adverse side effects, increased risk of infection or cancer.

In the preferred embod8iment, T-cells from the donor, an allogeneic or xenogeneic donor, will be cultured in vitro with recipient allogeneic or xenogeneic tissue which has been treated irradiated) to deplete host T-cells. To this culture, an effective amount of a gp39 antagonist, typically an anti-gp39 antibody, will be added 24-31 or 89-76 anti-human gp39 antibody will be added. This culture will be maintained for a time sufficient to induce T-cell tolerance. Typically, this time will range from about 1-2 days to 30 days, more typically about 5 15 days, and most typically about 10 days.

-11- After culturing, the donor T-cells can be tested to determine whether they elicit an anti-host allo- or xeno- response. Also, it can be determined whether such cells remain viable and otherwise elicit normal T-cell activity after treatment, IL-2 responses.

As shown in the Examples which follow, it has been found that donor T-cells when treated according to the invention exhibit markedly blunted antihost xeno- or alloantigen responses, maintained viability, and further maintain intact IL-2 responses. Also, upon restimulation, donor T-cells maintained their anti-host alloantigen hyperresponsiveness.

It was also observed that in the primary MLR, the production ofThelper Type 1(Th 1) cytokines was markedly reduced. Similarly, in secondary restimulation cultures, Thi cytokine production was also markedly reduced.

Moreover, it was found that in vivo administration of equivalent numbers of control or anti-gp39 (CD 154) monoclonal antibody treated donor T-cells had markedly different graft-versus-host disease properties.

Specifically, recipients of three-fold higher number of donor T-cells in controls had a 50% actuarial survival rate as compared to 0% in controls. In other experiments, up to a 30-fold difference in graft-versus-host disease potentials were observed using anti-gp39 (CD154) monoclonal antibody treated T-cells. Based thereon, we have surprisingly concluded that donor Tcells can be effectively tolerized ex vivo by a mixed lymphocyte reaction.

This should provide an important new approach for invoking donor T-cell tolerization to host cells and xenoantigens.

-12- The method provides significant potential in the area of bone marrow or peripheral blood cell transplantation therapies. Bone marrow and stem cell transplantation is conventionally utilized for treatment of various diseases, such as leukemia and other diseases involving immune cell deficiencies.

Moreover, bone marrow transplantation may afford benefits in the treatment of other diseases also, such as in the treatment of autoimmune diseases.

However, a prevalent risk associated with conventional bone marrow transplantation therapy is the risk of eliciting a GVHD response. The subject method should reduce or even eliminate such risk and thereby extend the clinical indications for bone marrow transplantation therapies.

Essentially, these methods will comprise treating bone marrow or peripheral blood cells ex vivo as described above, and introduction of the treated bone marrow or peripheral blood cells into a recipient in need of such treatment, a cancer patient or person suffering from an autoimmune disease, in need of immune reconstitution because their own lymphoid cells have been depleted as a result of the disease or treatment of the disease because of radiation treatment).

The present method may be combined with other anti-rejection treatments, in vivo infusion of immunosuppression agents such as methatrycide, cyclosporine A, steroids, or gp39 antagonist administration.

Ideally, the present method will provide for immune reconstitution in a recipient of the treated donor T-cells without eliciting any GVH response.

However, in some instances, this therapy may need to be repeated if the transplanted tissue does not "take" in the transplant recipient. Alternatively, it may be necessary if the lymphoid system of the transplant recipient becomes -13impaired again as a result of disease or treatment or the disease, e.g., subsequent radiation treatment. In such cases, suitable donor T-cells will again be contacted ex vivo with anti-gp39 antibody and T-cell depleted alloor xenoantigen bearing recipient cells, to induce T-cell tolerization, and then infused in the transplant recipient.

The invention is further illustrated by the following Examples which should not be construed as limiting. The contents of all references, patents and published patent applications throughout this application are incorporated by reference in their entirety.

EXAMPLE 1 The results of a mixed lymphocyte reaction (MLR) between donor CD4+ lymph node T cells and MHC Class II disparate alloantigen bearing stimulator cells is shown in Figure 1. In this experiment, highly purified CD4+ lymph node T cells from C.H2bm 1 2 were plated at a concentration of x 10 6 per ml final concentration in microtiter wells or in bulk culture in 24well plates. Stimulator cells were C57BL/6 T cell depleted, irradiated spleen cells used at a final concentration of 1 x 106 per ml. The MLR media consisted of 10% fetal calf serum, 5% supplements, and 2-ME. Anti-gp39 mAb was added at a final concentration of 50 micrograms per ml. Where indicated in Figure 1, IL-2 was added at a final concentration of 50 units per ml. Microtiter wells were pulsed with one microcurie per well of tritiated thymidine for an eighteen hour time period before harvesting. The mean A CPM (CPM of experimental CPM of responders alone) are shown on the y axis and the days of primary MLR culture on the x axis. These data P\OPER\Pk\2003261499 claims 350doc-21/12105 O -14demonstrate a profound hyporesponsiveness in anti-gp39 mAb treated cultures which is reversible by addition of exogenous IL-2.

EXAMPLE 2 Supernatants from vogue cultured cells from the experiment shown in Figure 1 were analyzed for the concentration of interleukin 2 These results are contained in Figure 1A. Supernatants were analyzed by ELISA (R&D Systems, Minneapolis, MN). Supematant concentration in pg per ml were shown on the y axis 1 and the days of MLR culture on the x axis. The addition of anti-gp39 mAb inhibited IL-2 production from donor T cells in a primary MLR culture.

EXAMPLE 3 The supernatant concentration of interferon gamma was analyzed by ELISA in the same cultures used in the experiment the results of which are contained in Figure 2A. These results are contained in Figure 2B. It can be seen that the addition of antigp39 mAb was observed to lead to a profound reduction of interferon gamma production and a primarily MLR culture.

EXAMPLE 4 At the end of the ten day cell culture period, cells were phenotyped by two color flow cytometry. As can be seen in Table 1 (after examples), the addition of antigp39 mAb did not prevent T cell activation as evidenced by the high levels of OX40, CTLA-4, B7-1 and B7-2. The addition of anti-gp39 mAb, however, did inhibit the conversion of naive T cells to effector T cells as demonstrated by the high levels of L-selectin, ICAM-I and low levels of CD45. Cells in the treated culture were not undergoing apoptosis that is evidenced by the relatively lower positivity for 7-AAD.

EXAMPLE At the end of the primary MLR culture, cells were washed and replated at a concentration of 3 x 104 per 96 well plate. To each well, irradiated splenocytes from C57BL6 mice were added at a concentration of 10' cells per well. These results are contained in Figure 3A. Where indicated, IL-2 is added at a final concentration of 50 units per ml. The media consisted of fetal calf serum, 5% supplements, 2-ME. Microtiter wells were labeled with one microcurie per well at the indicated times for a period of eighteen hours prior to harvesting. On the y axis are the mean proliferation values (A CPM) and on the x-axis are the days of secondary MLR culture. As can be seen from the results in Figure 3H, donor T cells exposed to anti-gp39 mAb in primary but not secondary culture retained alloantigen specific hyperresponsiveness in the secondary culture. This was reversible by the addition of exogenous IL-2 in the secondary culture alone.

EXAMPLE 6 In separate cultures, donor T cells from control treated cultures or antigp39 mAb treated primary MLR cultures were exposed to exogenous IL-2 at units per ml final concentration. These results are contained in Figure 3b.

It can be seen that there is an equivalent response of donor T cells from control treated as compared to anti-gp39 mAb treated primary MLR cultures as assessed under the secondary conditions.

EXAMPLE 7 Supernatants obtained from the secondary MLR bulk cultures were tested by ELISA for the production of IL-2 (Figure 4A) or interferon gamma (Figure 4B) as measured in a secondary MLR culture. It can be seen therefore -16that donor T cells exposed to anti-gp39 mAb in primary but not secondary MLR cultures continue to have a markedly low supernatant concentration of IL-2 (Figure 4A) and interferon gamma (Figure 4B).

EXAMPLE 8 At the end of the primary MLR culture, donor T cells were administrated to sublethally irradiated (600 cGray total body irradiation) C57BL/6 recipients. Two cell doses were tested (105 or 3 x 105). These results are contained in Figure 5. It can be seen from Figure 5 that recipients of controlled cultured cells at either cell dose uniformly succumb to lethal GVHD prior to four weeks post transplantation. In contrast, recipients of 105 donor T cells exposed to anti-CD40L mAb ex vivo had an 88% survival rate.

Recipients of 3 x 105 donor T cells exposed to anti-CD40L mAb had a survival rate of 50% at time periods greater than two months post transfer.

When compared to recipients of donor T cells obtained from control cultures, the actuarial survival rates of recipients of an equal number of donor T cells exposed to anti-CD40L mAb treated was significantly (p <0.001) higher at both cell doses.

EXAMPLE 9 The animals in the experiment, the results of which are contained in Figure 5A were monitored for evidence of GVHD by mean weight curves.

These results are contained in Figure 5B. It can be seen therefore that recipients of control cells had a marked decrease in mean weight curves (yaxis) beginning 2.5 weeks post transfer which resulted in GVHD lethality prior to 4 weeks post transfer. In contrast, recipients of anti-gp39 mAb treated cells had weight curves that exceeded their pre-transfer mean body -17weights. Also, recipients of 105 or 3 x 10' cells from control cultures had a marked reduction in mean body weight, consistent with a GVH reaction.

This demonstrated that GVHD lethality was inhibited by treatment with antigp39 mAb.

Moreover, in other experiments, up to a 30-fold reduction in GVHD mortality has been observed in recipients receiving anti-gp39 mAb treated cultures as compared to controls.

EXAMPLE The in vivo expansion of donor alloreactive T cells was examined.

Donor T cells from the experiment shown in Figures 1 5 and Table 1 and 2 were infused into mice with severe combined immune deficiency. These recipients were disparate with the donor at MHC class I class II loci. On day 6 post transfer, mice were given a continuous intravenous infusion of 1 ml per hour (representing about 1/4-1/3 of the animals total body water per hour) of fluids. The thoracic duct lymphatics were cannulated and thoracic duct lymphocytes were collected during an overnight collection procedure.

Approximately 1 ml per hour per animal is collected prior to death. The number of CD4+ T cells produced per day can then be quantified. It can be seen that the recipients of control cultured cells which produced an average of 2 x 106 CD4+ T cells per ml of thoracic duct effluent. By contrast, recipients of anti-gp39 mAb treated cultures produced only 0.3 x 106 CD4+ T cells per ml representing an approximate 7-fold reduction in the generation of alloreactive T cells in vivo. These data provide additional evidence that antigp39 mAb reduces the capacity of donor T cells in vivo to mediate lethal

GVHD.

-18- In summary, the results of the above experiments provide conclusive evidence that anti-gp39 mAb markedly reduced GVHD capacity in vivo. This represents a new methodology for tolerizing donor T cells to host antigens or alloantigens ex vivo as a means of preventing lethal GVHD in vivo.

Table 1: Exposure to Anti-CD4OL mAb, Ex Vivo In an MLR Culture Does Not Impair The Expression Of T Cell Activation Antigens Nor Induce Early Apoptosis But Does Inhibit The Conversion Of Naive T Cells To Effector Cells'.

CD4 CD25 0X40 CTLA-4 B7-1 137-2 L-selectin ICAM-1 CD45 7-AAD ICAM-1 CD4S 7-AAD Control 33 .15 28 39 51 33 45 92 100 (264) 100 (229) 100(493) 100(158) cagp39 mAb 58 48 23 'Cells at the end of the 10 day primary MLR culture were analyzed by 2-color FACS. The positive for the indicated molecules is listed. Activation antigens includes CD25, 0X40, CTLA-4, ]37-1, and ]37-2- Effector cell antigens include L-selectin, ICAM-1, and 7-AAD is an indicator of early apoptosis. The mean fluorescent channel is listed in Table 2: Exposure To Anti-CD40L mAb Ex Vivo In An MLR Culture Reduces the Expansion But Not Activation Of Donor T Cells In Nonirradlated Allogeneic Recipients'.

No. CD4+T CD4 T cells/ml. CD25 CD40L OX40 CTLA-4 B7- B7- L-selectin ICAM-1 CD45 1 2 CD44 7-AAD Non-BMT Ctrl Post-BMT Ctrl mAb 1.3 x 106 10 7 12 3 3 22 98(950) 70(76) 100 (375) 83 (215) 3 2.0 x 10 27 11 51 2 81 25 4(415) 100(295) 82(90) 72 45 12(522) 100(356) 100(103) 100 (1415) 7 0.3 x 10 6 20 5 52 3 86(1290) 'Thoracic duct lymphocytes were collected from normal donor strain controls 3) or from allogeneic SCID (severe combined immune deficient) recipients of control or anti-gp39 mAb treated cultures (cultures consisting of 2 mice and 3 mice were pooled due to low cell number and were then separately analyzed). Approximately 1 ml per mouse per hour is collected during an overnight cannulation procedure. Lymphocytes were analyzed by 2-color FACS. The mean positive for the indicated molecules is listed. When indicated, cells were gated for Cp4 positivity and then analyzed for the co-expression of the indicated antigen. Activation antigens include CD25, CTLA-4, B7-1, and B7-2. Effector cell antigens include L-selectin, ICAM-1, CD45 and CD44. 7-AAD is an indicator of early apoptosis.

The mean fluorescent channel is listed in P:\OPERPlkU2003261499 claims 330 doc-21/ II 2/05 Q- -21- Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

S 5 Throughout this specification and the claims which follow, unless the context ND requires otherwise, the word "comprise", and variations such as "comprises" and S"comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Claims (15)

1. A method for inducing T-cell tolerance or non-responsiveness of donor T-cells to alloantigens or xenoantigens ex vivo comprising the following: providing a culture containing donor T-cells; (ii) producing a mixed lymphocyte reaction culture by adding cells bearing n alloantigens or xenoantigens to said donor T-cell culture; S(iii) adding to the resultant mixed lymphocyte reaction culture of gp39 antagonist; and (iv) maintaining these cells in culture for about 10 to 30 days.

2. A method according to claim 1, wherein the donor T-cells are contained in a tissue -sample of donor bone marrow or peripheral bloods cells.

3. A method according to claim 1 or claim 2, wherein the gp39 antagonist is selected from the group consisting of an anti-gp39 antibody, soluble CD40, and soluble fusion protein.

4. A method according to claim 3, wherein the gp39 antagonist is an anti-gp39 antibody. A method according to claim 4, wherein the anti-gp39 antibody is an anti-human gp39 monoclonal antibody.

6. A method according to any preceding claim, wherein the cells bearing alloantigens or xenoantigens comprise cells or tissue obtained from a potential transplant recipient that has been treated to deplete recipient T-cells.

7. A method according to claim 6, wherein T-cell depletion is effected by irradiation.

8. A method according to any preceding claim, further comprising the step of P:\OPER\Pxk003261499 clams 350 doe-21/12/05 0 -23- U Sassaying the donor T-cells for elicitation of an anti-host response against alloantigens or Sxenoantigens.

9. A method according to claim 8, wherein the step of assaying comprises measuring interleukin-2 (IL-2) concentration in the cell culture medium supernatant of the donor T- IND cells. (N A method according to claim 9, wherein adding the gp39 antagonist inhibits IL-2 production.

11. A method according to claim 8, wherein the step of assaying comprises measuring interferon-gamma concentration in the cell culture medium supernatant of the donor T- cells.

12. A method according to claim 11, wherein adding the gp39 antagonist reduces interferon-gamma production.

13. A method according to claim 8, wherein the step of assaying comprises measuring the levels of L-selectin, ICAM-1, and CD45 on the donor T-cells.

14. A method according to claim 13, wherein adding the gp39 antagonist results in high levels of L-selectin, high levels of ICAM-1, or low levels of A method for preparing a therapeutic agent for treating a recipient in need of T-cell transplantation, comprising producing treated donor T-cells by a method as defined in any preceding claim and using the treated donor T-cells as an active ingredient in the therapeutic agent.

16. A method according to claim 15, wherein the recipient is in need of immune reconstruction as a result of disease or disease treatment. P:\OPER\Pxk\2003261499 clams 350 doc-21/I 2/05 -24- U

17. A method according to claim 16, wherein the disease is cancer or autoimmune disease.

18. A method for treating a recipient in need of T-cell transplantation, comprising administering to such recipient the therapeutic agent of claim (O 0 DATED this 21st day of December, 2005 REGENTS OF THE UNIVERSITY OF MINNESOTA and TRUSTEES OF DARTMOUTH COLLEGE by their Patent Attorneys DAVIES COLLISON CAVE