WO1991012849A1 - Engrafting allogeneic tissues or organs using interleukin - Google Patents

Abstract

Disclosed herein is a method for in vivo treatment of a mammal with interleukin 2 (IL-2) that protects against mortality from acute graft-versus-host disease (GVHD) caused by MHC mismatched lymphoid cells. Doses of 10,000 to 50,000 U of IL-2 twice daily for the first 5 days after allogeneic bone marrow transplant in lethally irradiated mice markedly reduced mortality from both acute and chronic GVHD induced across complete MHC barriers and frequently led to long-term survival. Complete allogeneic marrow reconstitution was demonstrated in all long-term survivors of this treatment. While administration of either IL-2 or T cell depleted (TCD) syngeneic marrow alone was protective in some experiments, maximal protection was observed by administering both IL-2 and TCD syngeneic marrow, especially when the effects of IL-2 were suboptimal. Timing of IL-2 administration was critical for protection, since a delay of seven days in commencing IL-2 treatment was associated with accelerated GVHD mortality. Anti-leukemic effects of allogeneic lymphocytes were not diminished by the co-administration of IL-2 and TCD syngeneic marrow.

Description

"ENGRAFTING ALLOGENEIC TISSUES OR ORGANS USING INTERLEUKIN".

FIELD OF THE INVENTION The present invention relates to methods for facilitating transplantation of allogeneic tissues or organs [i.e., where the major histocompatibility complex (MHC) and/or the minor histocompatibility determinants of the donor do not match those of the recipient] . In particular, this invention relates to a method for facilitating engraftment of an allogeneic tissue or organ using interleukin 2 (IL-2) , either alone or in combination with T cell depleted syngeneic bone marrow, to prevent graft-versus-host disease (GVHD) . BACKGROUND OF THE INVENTION

The phenomenon of alloreactivity has posed a long-standing challenge and obstacle to the field of clinical bone marrow and solid organ transplantation. Such reactivity is manifested by both GVHD and host- versus-graft responses in the form of graft rejection and resistance to subsequent alloengraftment. Nonspecific immunosuppression of the recipient has generally been used to control these phenomena. However, suppressive agents have their own associated toxicities, and GVHD and chronic graft rejection frequently persist despite their use. Therefore, attempts have been made to overcome the requirement for nonspecific immunosuppression in experimental animal models by manipulation of the host immune system with hemopoietic elements to induce donor specific transplantation tolerance.

One approach to achieving specific transplantation tolerance for any tissue or organ from a particular donor is to replace MHC-reactive elements of the immune system in the recipient with those from the donor. Thus, it has been found that elements of the immune system may be transferred permanently from an allogeneic donor by transplanting bone marrow into a recipient after ablation of the endogenous bone marrow and other hemopoietic elements (by lethal irradiation, for example) . Allogeneic bone marrow transplant (BMT) recipients are called chimeras because their hematopoietic cells are derived from the bone marrow of the donor which is antigenically different from the other recipient cells. The transplanted immune systems of these chimeras are fully tolerant of tissue or organ transplants from the donor of the allogeneic bone marrow. To avoid attack on host tissues by MHC- reactive immune system components of the donor, particularly certain T lymphocytes, which leads to GVHD, prior to implantation allogeneic bone marrow is often depleted of donor T cells by cell-specific cytocidal methods. While this approach does reduce the incidence of GVHD, unfortunately, there are two major problems being encountered with the clinical use of T cell-depleted bone marrow, particularly in the principal application of this therapy, the treatment of leukemia: 1) an increased rate of failure of marrow engraftment; and 2) an increased leukemic relapse rate, i.e., loss of the graft-versus-leukemia effect of the allogeneic T cells. These opposing benefits and drawbacks of using T cell depleted marrow have created the major dilemma currently impeding wider clinical use of bone marrow transplant.

In light of the frequent complications cited above that often lead to allograft failure or immunodeficiency, bone marrow transplantation in humans generally has been confined to treatment of acute life- threatening conditions, such as certain leukemias or severe bone marrow depletion (e.g., by accidental irradiation) , where replacement of the bone marrow is the only effective therapy. To ensure reasonable prospects for successful transplantation, the MHC profiles of the recipient patient and the donor must be closely matched to reduce the chances of graft rejection and GVHD. Consequently, in this country alone, thousands of BMT candidates die each year for want of an acceptable MHC-matched donor.

Furthermore, in the case of leukemia patients, the largest group of BMT candidates for current procedures, even when BMT is successful with respect to engraftment of the marrow, the treatment may fail to eliminate the cancer. Evidently, pretransplantation ablation of the recipient hemopoietic system often leaves some viable leukemic cells in the body which may then be eliminated by the transplanted immune system. Failure of the transplant to regenerate full immune functionality thus may lead to resurgence of the leukemia. In particular, even without deliberate T cell depletion, the donor bone marrow may not contain sufficient numbers of certain T cells that are critical for providing full anti-leukemic activity in the transplanted immune system. Thus, T cell depletion of bone marrow prior to transplantation often reduces the effectiveness of this treatment for leukemia patients. Interleukin-2 (IL-2; originally known as T cell growth factor) is a soluble protein factor having lymphopoietic cell-specific regulatory capacities. IL- 2 is known as a cytokine, and more particularly as a lymphokine, owing to its production by lymphocytes. IL-2 has been found to play a variety of important roles in regulating the responsiveness of the immune system. The availability of.,large amounts of purified IL-2 from specialized human cell cultures or, more recently, from recombinant DNA systems, has facilitated considerable research on the potential therapeutic utility of L-2. Findings of deficiencies of IL-2 and of certain IL-2-responsive T cells in reconstituted immune systems of BMT patients have led to suggestions of the possibility of using L-2 to facilitate bone marrow transplantation (see, for example, Welte, K. , Ciobanu, N. , Moore, M.A.S., Gulati, S. O'Reilly, R. J. , and Mertelsmann, R., 1984, Blood 64:380-85: and U.S. Patent 4,778,879 to R. Mertelsmann et al., issued October 18, 1988). Indeed, several in vitro studies have shown promising stimulatory effects of IL-2 on isolated T cells from human or animal BMT recipients. Unfortunately, in vivo experiments have shown that administration of IL-2 to allogeneic BMT recipients can induce a dramatic potentiating effect on GVHD, unless the marrow is depleted of T cells before transplantation. Thus, for example, Jadus and Peck, 1983, found in mice receiving untreated allogeneic bone marrow, that four doses of purified mouse IL-2, each consisting of ten units, given during the first six days after transplantation, increased mortality due to GVHD, from 60-80% to 100% (Jadus, M.R. , and A.B. Peck. 1983. Lethal urine graft-versus-host disease in the absence of detectable cytotoxic T lymphocytes. Transplantation 36:281-89). In contrast, using a different mouse model for allogeneic BMT in which the level of GVHD was low (less than 15% of the mice in the untreated control group died), Merluzzi et al., 1985, were unable to detect any enhancement of mortality from GVHD by recombinantly produced human IL-2 (rIL-2) at 1000 units per mouse three times per week for the first four weeks after transplantation (Merluzzi, V. J. , Welte, K., Last-Barney, K. , Mertelsmann, R. , et al., 1985, _ Immunol. 134:2426-2430) . These authors remained convinced that complete dose-curve analyses of IL-2 in vivo would be needed to answer the question of whether IL-2 aggravates GVHD. Subsequently, Malkovsky, et al., 1986, did find that administration of higher doses of human rIL-2 substantially increased mortality from GVHD in mice receiving conventional allogeneic marrow transplants, using a regimen of 2000 units human rIL-2 per mouse, given three times per week for five weeks after transplantation; however, the same treatment had no effect on animals reconstituted with T cell depleted allogeneic marrow and spleen cells

(Malkovsky, M. , M.K. Brenner, R. Hunt, S. Rastan, C. Dore, S. Brown, M.E. North, G.L. Asherson, H.G. Prentice, and P.B. Medawar. 1986. T-cell depletion of allogeneic bone marrow prevents acceleration of graft- versus-host disease induced by exogenous interleukin 2. Cell. Immunol. 103:476) . More recently, Sprent, et al., 1988, using selected fractions of allogeneic T cells to induce GVHD in mice, found that still higher doses of rIL-2 dramatically accelerated GVHD mortality, as indicated by a marked increase in the rate at which mortality from GVHD reached 100%, when a total of six doses of human rIL-2 were given, at 5000 units per dose, with a single dose given every other day beginning seven days after allogeneic T cell transplant (Sprent, J., M. Schaefer, E. Gao, and R. Korngold. 1988. Role of T cell subsets in lethal graft-versus- host disease (GVHD) directed to class I versus class II H-2 differences. I. L3T4^* cells can either augment or retard GVHD elicited by Lyt-2⁺ cells in class I- different hosts. J. EXP. Med. 16_7:556-569) . Thus, the prior art on the use of IL-2 in bone marrow transplantation teaches that administration of high doses of this multifunctional cytokine after transplantation of allogeneic bone marrow aggravates GVHD, particularly acute forms of this disease, unless the allogeneic marrow is depleted of T cells prior to transplantation.

The present inventors have recently demonstrated a new approach to induction of specific transplantation tolerance across major MHC barriers, as evidenced by specific acceptance of donor-type skin grafts. This method involves the use of syngeneic T cell depleted (TCD) bone marrow to ameliorate GVHD. Efficacy has been demonstrated by reconstitution of lethally irradiated mice with syngeneic TCD marrow in conjunction with untreated allogeneic bone marrow (Ilstand, S. T., et al., 1986, J. Immunol. 136:28-33). In this model system, transplantation of syngeneic TCD marrow with untreated allogeneic marrow could be accomplished without evidence of immediate GVHD or immunoincompetence. However, when allogeneic spleen cells were also transplanted with the BMT under the same conditions, which provided a more potent T cell challenge to the protective effects of the syngeneic TCD marrow, mortality from acute GVHD was retarded but not eliminated by the syngeneic TCD bone marrow. In further studies it was found that delaying the transplantation of allogeneic marrow and spleen cells for eight days after the syngeneic TCD marrow transplant enhanced effectiveness against GVHD (Sykes, M. , et al., 1988, Transplantation 46:327-330).

Nevertheless, the magnitude of the protection from syngeneic TCD bone marrow alone against acute GVHD mortality is limited, and no reduction of eventual mortality from chronic GVHD has been evident as a result of prior or simultaneous transplantation of syngeneic TCD bone marrow with allogeneic marrow and spleen cells. Furthermore, this method of using syngeneic TCD bone marrow obviously is not applicable in those cases where for any reason healthy syngeneic marrow is not available from the transplant recipient. Accordingly, avoiding GVHD while retaining the engraftment-promoting and anti-leukemic effects of T cells in allogeneic marrow remains a major challenge in the field of bone marrow transplantation.

SUMMARY OF THE INVENTION This invention is based on the discovery by the present inventors that administration in vivo of IL-2 immediately after transplantation of lymphocyte- enriched (with spleen cells) allogeneic bone marrow in lethally irradiated mice protected against GVHD mortality from allogeneic lymphocytes. Dose rates of IL-2 effective for providing protection against GVHD according to the present method approach the upper limit of tolerance of IL-2 for the transplant recipient and may vary from one individual to another within a given species, depending, inter alia, on previous therapy (e.g., radiation treatment) and basic health condition. In the present mouse model, effective dose rates ranged from about 4 x 10⁵ units to about 2 x 10⁶ units per kilogram of body mass per 0.5 day._: IL-2 treatments at these rates for durations of from about 2.5 to 5 days were effective for providing protection against GVHD. In contrast, delaying administration of the same IL-2 treatments until one week after allogeneic bone marrow transplantation resulted in acceleration of GVHD mortality, as taught in the prior art (Sprent, J. , M. Schaefer, E. Gao, and R. Korngold, 1988, supra). IL-2 treatment that protected against GVHD did not inhibit complete allogeneic repopulation of the lymphopoietic systems of most transplant recipients, nor did it diminish the anti-leukemic effects of allogeneic lymphocytes. Furthermore, the anti-GVHD efficacy of this IL-2 treatment in such transplant procedures was increased by combination with the known anti-GVHD effect of co-transplantation of TCD syngeneic bone marrow with the allogeneic marrow. For suboptimal amounts of IL-2, maximal protection from GVHD was achieved when TCD syngeneic marrow was also administered. Survivors protected from GVHD with this combined treatment also demonstrated complete allogeneic lymphopoietic repopulation, as did the lymphopoietic systems of similar but less frequent chimeric survivors of this allograft procedure having no IL-2 treatment. Such fully allogeneic lymphopoietic systems are known to exhibit full tolerance upon subsequent tissue or organ transplantation from the donor of the allogeneic lymphopoietic cells. Accordingly, the present invention relates to a method for facilitating engraftment of allogeneic bone marrow in a mammal comprising administration of interleukin 2 to that mammal beginning about the time of marrow transplantation, wherein the dose rate and duration of IL-2 administration is effective to provide protection against graft-versus-host disease. The IL-2 may be derived from any mammalian cell or tissue source or recombinant DNA production system. (See, for examples, U.S. Patent 4,840,934 to Anderson, issued June 20, 1989; and Hank, J.A., Kohler, P.C., Weil- Hillman, G. , et al., 1988, In vivo induction of the lymphokine-activated killer phenomenon: interleukin 2- dependent human non-major histocompatibility complex- restricted cytotoxicity generated in vivo during administration of human recombinant interleukin 2. Cancer Res. 48:1965-71.) Advantageously, the IL-2 is of the same mammalian species as the transplant recipient to be treated with the IL-2. The IL-2 may be administered by any method which is convenient and effective, for example, by injection or by infusion according to regimens known in the art for administration of IL-2 for other purposes. (See, for examples. Hank, J.A., 1988, supra; M. Syman, A. Bosly, C. Gisselbrecht, P. Price, C. Franks, 1989, Cancer Treatment Rev. 16 (Sup. A):15-19; Rosenberg, S. A. et al., 1989, Ann. Surer.. 210:474-485; S.A. Rosenberg, J.J. Mule, P.J. Spiess, CM. Reichert, S.L. Schwarz, 1985, J. Exp. Med. 161:1169; Papa, M. Z. et al., 1988, Cancer Res. 48:122-129; and Kolitz, J. E., Wong G. Y., Welte, K. , Merluzzi, V. J. , Engert, A., Bialas, T. , Polivka, A., Bradley, E.C., Konrad, M. , et al., 1988. Phase I trial of recombinant interleukin-2 and cyclophosphamide augmentation of cellular immunity and T-cell mitogenic response with long-term administration of rIL-2. J. Biol. Response Modif. 2:457-472.) According to one embodiment of this aspect of the invention, the IL-2 is administered in the-form of multiple discrete uniform doses at regular intervals. Preferably, the first of these doses is administered within the period extending inclusively from about three hours before marrow transplantation to about one hour after transplantation, and subsequent doses are administered approximately every twelve hours thereafter.

In the above method of this invention as applied to a mouse model of mammalian physiology in experiment described hereinbelow, IL-2 treatments effective for providing protection against GVHD were administered according to the above schedule in ive or ten uniform doses over a total period of about 2.5 days or 5 days; a single dose given at the time of transplantation failed to protect against GVHD. Uniform dose rates of IL-2 that were found to be effective for providing protection of a mouse against GVHD ranged from about 10,000 to 50,000 units of recombinant human IL-2 every twelve hours for a body mass on the order of 0.025 kilogram, corresponding to a dose rate range of from about 4 x 10⁵ to about 2 x 10⁶ units of IL-2 per kilogram of body mass per 0.5 day. Accordingly, the present invention particularly relates to the method of facilitating engraftment of allogeneic bone marrow in a mammal, as described above, in which the total number of uniform doses of interleukin 2 is greater than one but not more than ten doses. Further, each of these uniform doses advantageously consists essentially of from about 4 x 10^s to about 2 x 10⁶ units of interleukin 2 per kilogram of body mass. In the present mammalian model, the largest tested amount of IL-2 (10 doses of 50,000 units) was most effective for providing protection against GVHD but was also found to cause symptoms indicative of cumulative toxicity of IL-2. Reducing the amount of IL-2 administered by halving the number of 50,000-unit doses produced a shortened treatment course of about two and one half days that was at least as protective against GVHD mortality as was a full five-day course; and the animals showed no clinical evidence of adverse effects. Thus, in this particular mammalian system, toxicity of the IL-2 treatment was reduced without reducing effectiveness of the protection against GVHD by maintaining the highest tolerable dose rate but reducing the duration of the treatment. It will be recognized by one skilled in the art that determination of the dose rate and duration of IL-2 administration that is effective to provide protection against graft-versus-host disease for any given mammalian species is straightforward, according to the biological and statistical methods that have been applied to the animal model as described hereinbelow. Thus, in general, the upper limit of tolerance of IL-2 dose rate is determined based on symptoms of IL-2 toxicity which are characteristic of the selected species (e.g., see Hank, J.A. , Kohler, P.C., Weil-Hillman, G. , et al., 1988, supra). Similarly, symptoms of GVHD are well known in the art (e.g., see the Detailed Description, below; C. Hershko and R. P. Gale, 1980. GVHD scoring systems for predicting survival of specific mortality in bone marrow transplant recipients. In Gale, R.P., Fox, C. F.,_-(eds.) Biology of Bone Marrow Transplantation, Academic Press, New York, pp 59-68; and H. Glucksberg, R. Storb, A. Fifer et al., 1974. Clinical manifestations of graft-vs-host-disease in human recipients of marrow from HLA-matched sibling donors. Transplantation .18.:295-304) . Once the maximum tolerable dose rate of IL-2 has been established for a mammalian species, the duration of treatment effective against GVHD is determined by monitoring for symptoms of GVHD. The most critical parameters of administration of IL-2 with respect to the possibility of aggravating rather than preventing GVHD is failure to initiate treatment soon enough after transplantation and at a sufficiently high dose rate. Subsequent treatment with IL-2 after an initial course which is effective against GVHD does not aggravate GVHD.

Therefore, toxicity of IL-2 rather than concern for the possibility of aggravation of GVHD is the limiting factor in determining the maximum duration of IL-2 treatment that is efficacious in the facilitation of transplantation according to the method of the present invention.

According to another embodiment of the present invention, the amount of IL-2 needed for protection against GVHD can be reduced by co-transplantation of TCD syngeneic bone marrow with the allogeneic marrow. Thus, the present invention also relates to the method of facilitating engraftment of allogeneic bone marrow described above, further comprising transplanting T cell depleted syngeneic bone marrow to the mammal prior to or during transplantation of the allogeneic bone marrow. In the mouse model below, the two marrow specimens are admixed and cotransplanted; in a clinical setting, however, one skilled in the art would recognize that practical considerations might require the two marrow specimens to be administered at different times. Preferably the syngeneic marrow is transplanted first and may be transplanted at least eight days prior to transplantation of the allogeneic marrow. In any case, IL-2 treatment is initiated at about the time of transplantation of the allogeneic marrow. This marrow combination method obviously is only applicable in those cases where viable syngeneic marrow is available from the intended recipient of the allogeneic transplant.

In experiments described below, the present inventors have shown that the protective effect of IL-2 or IL-2 in combination with TCD syngeneic bone marrow is sufficient to prevent GVHD, which is caused by allogeneic T cells, even when allogeneic mouse bone marrow is supplemented with additional T cells from the donor spleen. However, it should be noted that in the mouse model, the level of T cells introduced by way of the allogeneic bone marrow alone is not nearly as high as T cell levels obtained by the usual process of harvesting human bone marrow for transplantation. The human method results in greater enrichment of the marrow with T cells from contaminating blood than in mouse bone marrow that is harvested surgically. Thus, addition of spleen cells to mouse marrow is necessary

SUBSTITUTE SHEET to achieve levels of allogeneic T cells in the mouse transplant that are comparable to T cell levels in human marrow. Yet the present inventors have shown that, with IL-2 treatment according to the present -method, supplementing the allogeneic mouse marrow with allogeneic lymphopoietic (spleen) cells advantageously enhances the immunocompetence of the repopulated lymphopoietic system, as in treatment of leukemia, for example. Therefore, notwithstanding the fact that, absent T cell depletion of human marrow, such marrow is in effect inherently supplemented with donor T cells, where it is desirable, human marrow can be supplemented with additional donor T cells (peripheral blood lymphocytes, for instance) according to the method of the present invention. Accordingly, the present invention also relates to the method of facilitating engraftment of allogeneic bone marrow in a mammal by administering IL-2, as described above, further comprising the transplantation of allogeneic lymphopoietic cells in addition to the allogeneic bone marrow. It will be understood by one skilled in the art of transplantation, of course, that allogeneic lymphopoietic cells are ordinarily contained in human marrow and thus marrow and lymphopoietic cells are obtained from a single donor.

The present inventors have demonstrated previously that survivors protected from GVHD by TCD syngeneic marrow without IL-2 treatment also show complete lymphopoietic repopulation with allogeneic cells, as do the similar chimeric survivors of the IL-2 treatment, and that such fully allogeneic lymphopoietic systems exhibit full tolerance upon subsequent tissue or organ transplantation from the donor of the allogeneic lymphopoietic cells (Ildstad, S.T., S.M. Wren, J.A. Bluestone, S.A. Barbieri, D. Stephany, and D.H. Sachs. 1986. Effect of selective T cell depletion of host and/or donor bone marrow on lymphopoietic repopulation, tolerance, and graft-vs-host disease in mixed allogeneic chimeras (BIO + B10.D2—>B10) . J. Immunol. 136:28) . Therefore, in another aspect, the present invention also relates to a method for facilitating engraftment of an allogeneic organ or tissue other than bone marrow in a mammal in which allogeneic bone marrow cells and, optionally, additional allogeneic lymphopoietic cells from the proposed organ or tissue donor are first engrafted into that mammal by administering to that mammal, within the period from about three hours before to five days after marrow transplantation, an effective amount of interleukin 2 to provide protection against graft- versus-host disease, whereby said mammal becomes tolerant to organ or tissue transplants from said donor. Thus, the methods of this invention are of general utility for the facilitation of transplantation of any allogeneic organ or tissue.

* * * The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples and Figures described therein.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Effect of IL-2 and TCD syngeneic marrow on GVHD mortality from A/J lymphocytes. Lethally irradiated BIO mice received 8xl0⁶ A/J spleen cells plus 15xl0⁶ A/J BMC, with or without 5xl0⁶ TCD BIO BMC and IL-2, 50,000 U i.p. twice daily for five days. Panel A: Survival of control animals not receiving A/J spleen cells; * TCD BIO BMC alone (n=5) ; • TCD BIO BMC plus IL-2 (n=5) ; Q TCD BIO plus A/J BMC (n=5) ;

Δ TCD BIO BMC plus A/J BMC plus IL-2 (n=5) ; 0 A/J BMC (n=2) ; A A/J BMC plus IL-2 (n=3) . In panels B, C, and

D, indicates the survival of animals receiving only

A/J BMC plus spleen cells (n-15) . Panel B: Survival curve of animals receiving A/J BMC plus spleen cells plus TCD B10 BMC (n=15) . Panel C: Survival curve of animals receiving A/J BMC plus spleen cells plus IL-2 (n=15) . Panel D: Survival curve of animals receiving A/J BMC plus spleen cells plus TCD B10 BMC plus IL-2 (n=14) .

Figure 2: Effect of IL-2 and TCD syngeneic marrow on rapid, acute GVHD mortality produced by A/J lymphocytes. Lethally irradiated B10 mice received 9xl0⁶ A/J spleen cells plus llxlO⁶ A/J BMC, with or without 5xl0⁶ TCD B10 BMC and IL2, 10,000 U twice daily for 5 days. Panel A: Survival of control animals not receiving A/J spleen cells; * TCD B10 BMC plus IL-2 (n=3) ; O TCD B10 BMC plus A/J BMC (n=3) ; ^ TCD B10 BMC plus A/J BMC plus IL-2 (n=5) . In panels B, C, and D, indicates the survival of animals receiving A/J

BMC and spleen cells alone (n=10) . Panel B:

Survival curve of animals receiving A/J BMC plus spleen cells plus TCD B10 BMC (n=9) . Panel C: Survival curve for animals receiving A/J BMC.plus spleen cells plus IL-2 (n=9) . Panel D: Survival curve for animals receiving A/J BMC plus spleen cells plus TCD B10 BMC and IL-2 (n=9) .

SUBSTITUTE SHEET Figure 3: IL-2 alone prevents acute GVHD mortality, but maximal early survival is achieved in recipients of TCD syngeneic marrow plus IL-2. Top and bottom panels: two independent experiments showing survival in lethally irradiated mice reconstituted with similar inocula containing A/J BMC plus A/J spleen cells, along with: no additional treatment ; TCD syngeneic marrow co-administered in the reconstituting inoculum on day 0 ---- ; IL-2, 50,000 U twice daily on day 0-4 (top) or day 0-2 (bottom) plus TCD syngeneic marrow co-administered in the reconstituting inoculum on day 0 . Each group contained 8 to 10 animals.

Control animals not receiving A/J spleen cells demonstrated excellent survival in both experiments. Figure 4: Effect of timing of IL-2 administration on GVHD mortality. Lethally irradiated B10 mice received 5xl0⁶ TCD B10 BMC, 10x10⁶ A/J BMC, and

9xl0⁶ A/J spleen cells. Survival of control animals not receiving IL-2 (n=10) ; survival of animals receiving 10,000 U IL-2 twice daily for 5 days beginning on the day of BMT (n=10) ; - - - - survival of animals receiving 10,000 U of IL-2 twice daily for 5 days beginning 7 days after BMT (n=10) . Control animals (not shown; n=5) receiving A/J marrow plus TCD syngeneic marrow demonstrated 100% survival.

Figure 5: Assessment of the number of IL-2 doses required for protection against GVHD mortality. Lethally irradiated B10 mice received 5 X 10⁶ TCD BIO BMC, lOxlO⁶ A/J BMC, and 9xl0⁶ A/J spleen cells. Survival of control animals receiving no IL-2 (n=10) ; — • • — survival of animals receiving a single dose of 50,000 U IL-2 immediately prior to BMT (n=9) ; survival of animals receiving

50,000 U IL-2 twice daily for 2.5 days (i.e. 5 doses), beginning immediately prior to BMT (n=8) ; survival of animals receiving 50,000 U IL-2 twice daily for 5 days (i.e. 10 doses) beginning immediately prior to BMT (n=9) . Control animals (not shown) receiving TCD syngeneic marrow along (n=5) or with IL-2, 50,000 U twice daily for 5 days (n=5) , demonstrated 100% survival.

Figure 6: Examples of the phenotype of lymphopoietic cells repopulating lethally irradiated B10 mice treated or not treated with IL-2, 50,000 U twice daily for 5 days beginning on the day of BMT. B10 mice were lethally irradiated and reconstituted with either TCD B10 BMC plus B10.D2 BMC, or with TCD B10 BMC, B10.D2 BMC and B10.D2 spleen cells, or with B10.D2 BMC and spleen cells alone, as indicated. PBL were obtained 15 weeks after BMT, stained with mAbs, and analyzed using FCM, as indicated in the Materials and Methods section. Staining with K^b-specific mAb 5F1 ; staining with D^d-specific mAb 34-2-12 .

Figure 7: A. Survival of lethally irradiated B10 mice receiving intravenous inocula containing: TCD

B10 BMC plus EL4 leukemia cells ( ;n=4) ; TCD B10

BMC plus EL4 leukemia cells and i.p. IL-2 ( ;n=4) ; A/J BMC, A/J spleen cells, EL4 leukemia cells, and i.p.

IL-2 ( ;n+9) ; TCD B10 BMC, A/J BMC, A/J spleen cells, EL4 leukemia cells, and i.p. IL-2 (^*—^*~"";n=10) . B. Survival in the same experiment of lethally irradiacted BIO mice receiving intravenous inocula containing: A/J BMC plus A/J spleen cells (

;n=9) ; A/J BMC plus A/J spleen cells, and i.p. IL-2 ( ;n=10) ; TCD BIO BMC, A/J BMC plus A/J spleen cells, and i.p. IL-2 ( —- ;n=10) .

Figure 8: A. Survival of lethally irradiated BIO recipients of: TCD BIO BMC plus^* EL4 cells and i.p. IL-2 ( ; n-10) ; TCD BIO BMC, EL4 cells, plus A/J BMC ( ; n-10) ; TCD BIO BMC, EL4 cells, A/J BMC and

A/J spleen cells, and i.p. IL-2 ( - - - - ;n-10) . B. Survival of recipients in the same experiment of: A/J

BMC plus A/J spleen cells ( ; n=9) ; A/J BMC plus

A/J spleen cells plus EL4 cells ( ; n=10) ; A/J BMC, A/J spleen cells, plus TCD BIO BMC *~-; n=10) ; A/J BMC, A/J spleen cells, EL4 cells, and i.p. IL-2 ( — • •

- ; n=10) ; A/J BMC, A/J spleen cells, TCD BIO BMC, EL4 cells, and i.p. IL-2 ( - - - - ; n=10) .

Figure 9: Survival of lethally irradiated BIO recipients of: TCD BIO BMC, EL4 cells, and i.p. IL-2 ( ; n-10) ; TCD BIO BMC, 30xl0⁶ A/J BMC, plus EL4 cells

( ; n=9) ; TCD BIO BMC, 30xl0⁶ A/J BMC, EL4 cells. and i.p. IL-2 ( ; n-10) ; TCD BIO BMC and 30xl0⁶

A/J BMC (^—'—; n-4); TCD BIO BMC, 30xl0⁶ A/J BMC, and i.p. IL-2 ( — • • — ; n-4).

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention relates to a method for facilitating engraftment of allogeneic bone marrow in a mammal by administering to that mammal, within the period from about three hours before to five days after marrow transplantation, an effective amount of IL-2 for providing protection against GVHD. This section

SUBSTITUTE SHEET describes the details of the experiments which demonstrate that IL-2 administered in vivo at the time of BMT has a potent effect in preventing mortality due to both acute and chronic GVHD. Furthermore, evidence is presented that the combination of IL-2 and TCD syngeneic marrow provides optimal protection against acute GVHD mortality. Neither IL-2 alone nor IL-2 plus TCD syngeneic marrow prevented complete lymphopoietic reconstruction by co-administered allogeneic BMC plus spleen cells. This novel method of achieving complete allogeneic lymphopoietic repopulation while reducing GVHD mortality presents a new approach to solving the opposing problems of GVHD versus failure of alloengraftment associated with T cell depletion for the prevention of GVHD. Finally, the observation that administration of IL-2 still permits the anti-leukemic effects of allogeneic T cells is demonstrated in a murine leukemia model which has recently been described (Sykes, M. , Z. Bukhari, and D.H. Sachs. 1989. Graft- versus-leukemia effect using mixed allogeneic bone marrow transplantation. Bone Marrow Transplant 4.:465.) .

""Previous reports from the present inventors demonstrated that TCD syngeneic marrow can delay mortality from acute GVHD (Ildstad, S.T., et al., 1986, supra) . In the experiments reported here, a protective effect of TCD syngeneic marrow against acute GVHD has been detected only when the GVHD was mild in severity (e.g., Figure 1). Furthermore, consistent with the previous report (Ildstad, S.T., et al., 1986, supra), TCD syngeneic marrow alone did not prevent late mortality from chronic GVHD. Thus, TCD syngeneic marrow alone has a limited ability to prevent acute GVHD mortality and no detectable effect on chronic GVHD. In contrast, the addition of high doses of IL-2 (from 10,000 to 50,000 units twice daily for 2.5 to 5 days for a body mass on the order of 0.025 kilogram) leads to increased protection from acute GVHD mortality (e.g.. Figures 2, 4, 5), as well as significant protection from chronic GVHD mortality (e.g.. Figure 1) . Protection from chronic GVHD mortality is apparent regardless of whether or not TCD syngeneic marrow is co-administered.

In the absence of TCD syngeneic marrow, IL-2 also has significant protective activity against GVHD mortality, but, in every instance, such protection was increased when TCD syngeneic marrow was co-administered (e.g.. Figures 2, 3). The capability of TCD syngeneic marrow to increase the protective effect in recipients of IL-2 was most apparent in experiments in which IL-2 alone provided sub-optimal protection (e.g.. Figure 2). In some experiments, the degree of protection afforded by IL-2 alone was so potent that there was little room for improvement by the addition of TCD syngeneic marrow (e.g., Figure 3). The reasons for the variability in the degree of protection afforded by similar doses of IL-2 alone are as yet unclear. As discussed in the Summary, above, if this model were to be applied to larger animals or man, it is possible that IL-2 toxicity would prevent administration of optimal doses, in which case the improved protection provided by the addition of TCD autologous marrow might be desirable where this option is available.

SUBSTITUTE SHEET The results below appear to conflict with those of other workers, who have found GVHD is potentiated, rather than abrogated, by in vivo administration of IL-2 (Sprent, J., M. Schaefer, E. 5. Gao, and R. Korngold. 1988. Role of T cell subsets in lethal graft-versus host disease (GVHD) directed to class I Versus class II H-2 differences I.L3T4+ cells can either augment or retard GVHD elicited by Lyt-2+ cells in class I-different hosts. J. Exp. Med. 167:556- 0 569; Malkovsky, M., M.K. Brenner, R. Hunt, S. Rastan, C. Dore, S. Brown, M.E. North, G.L. Asherson, H.G. Prentice, and P.B. Medawar. 1986. T-cell depletion of allogeneic bone marrow prevents acceleration of graft- versus-host disease induced by exogenous interleukin 2. 5 Cell. Immunol. 103:476; Jadus, M.R. , and A.B. Peck. 1983. Lethal murine graft-versus-host disease in the absence of detectable cytotoxic T lymphocytes. Transplantation 36:281). This discrepancy could be due to several differences in the systems studied, 0 including the fact that these workers did not co- administer TCD syngeneic marrow, administered lower doses of IL-2, and used a delayed or prolonged time course of IL-2 administration. Consistent with this possibility, protection was observed when IL-2 was administered for 5 days starting on day 0, but acceleration of mortality occurred when administration of the same dose was begun after a 7-day delay.

In one of five dose-response titrations in the A/J into BIO strain combination, greater protection was provided by decreasing, rather than increasing doses of IL-2. Although there is no definite explanation for this variability in IL-2 toxicity between experiments, these results, the apparent illness of some animals after the third day of high-dose IL-2 treatment, and the observation that IL-2 therapy beginning on day 7 leads to accelerated mortality, suggested that a shorter course of high-dose IL-2 might optimize survival by limiting IL-2 toxicity. The results of such an approach (Figure 5) support this notion, and a short-term high-dose protocol may prove to be optimal for avoiding both GVHD and IL-2 toxicity. In summary, the present application discloses a new approach to the problem of preventing mortality from acute and chronic GVHD which does not prevent alloengraftment and does not require T cell depletion of allogeneic bone marrow. This depletion is associated with an increased incidence of failure of alloengraftment, increased probability of leukemic relapse (P.J. Martin, J.A. Hansan, B. Torok-Storb, et al., 1988, Bone Marrow Transplant 3:445; N.A. Kernan, N. Flomenberg, B. Dupont, R.J. O'Reilly, 1987, Transplantation 43.:842; K.M. Sullivan, P.L. Weiden, R. Storb, et al., 1989, Blood 73:1720; P.L. Weiden, K.M. Sullivan, N. Flournoy, R. Storb, E.D. Thomas, 1981, New Enql. J. Med. 3_04:1529), and delayed recovery of T lymphocyte functions (CA. Keever, T.N. Small, N. Flomenberg, et al.. Blood 23:1340). IL-2 appears to be the first agent without known immunosuppressive properties with anti-GVHD activity. This observation prompted attempts to apply the present invention in a murine leukemic model. Thus, additional experimental results below demonstrate a significant anti-leukemic effect of allogeneic marrow and spleen cells in animals which were simultaneously protected against GVHD by administration of high dose IL-2 plus TCD syngeneic marrow. Furthermore, all surviving animals demonstrated complete allogeneic reconstitution of peripheral blood lymphocytes, regardless of whether or not they had received TCD syngeneic marrow and leiikemic (EL4) cells in addition to IL-2 (data not shown) . IL-2 plus TCD syngeneic marrow therefore reduces GVHD mortality while permitting an anti-leukemic effect and engraftment of allogeneic cells.

Whatever the mechanism, the present results suggest that high-dose IL-2 may have the potential to overcome the clinical dilemma whereby allogeneic T cells are essential for optimal anti-leukemic effects and for optimal alloengraftment, but produce the undesirable consequence of GVHD. T cell depletion, which remains the most effective known method of abrogating GVHD in human BMT recipients, has been associated with an increased probability of leukemic relapse in several hematologic malignancies. Evidence has also been obtained that the widely utilized immunosuppressive agent, cyclosporin, may also increase leukemic relapse probability (K. Atkinson, J.C Biggs, A. Concannon, A. Dodds, 1989, Aust. N. Z. J. Med. 4.:587). IL-2 appears to represent the first agent for abrogating GVHD which lacks known non-specific immunosuppressive properties. On the contrary, IL-2 is known to be capable of shrinking solid tumors in humans and animals (S.A. Rosenberg, M.T. Lotze, J.C. Yang, et al, 1989, Ann. Surq. 210:474; S.A. Rosenberg, J.J.

Mule, P.J. Spiess, CM. Reichert, S.L. Schwarz, 1985, . Exp. Med. 161:1169; T.D. Anderson, T.J. Hayes, M.K. Gately, J.M. Bontempo, L.L. Stern, G.A. Truitt, 1988, Lab. Invest. 59:598), and recent evidence also suggests that IL-2 may have anti-leukemic effects [S. Slavin, A. Eckerstein, L. Weiss, Nat. Immun. Cell Growth Reσul. 2:180 (1988); N.E. Kay, M.M. Oken, J.J. Hazza, E.C. Bradley, Nouv. Rev. Fr. Hematol. 30:475 (1988)].' The small anti-leukemic effect of IL-2 alone against the EL-4 tumor used here does not rule out the possibility of a stronger anti-leukemic effect against other hematologic malignancies, since tumors vary in their in vivo sensitivity to IL-2.

Further attempts to understand the mechanism of this novel anti-GVHD effect of IL-2 will help to determine its ultimate potential for clinical application. Additional details of the present experimental work are presented below.

Materials and Methods Animals: Male and female C57BL/10SnJ (BIO, H- 2^b, I^I^D¹³) , B10.D2/nSn (B10.D2, H-2^d, I^I^V) , and A/J (H-2^a, K^kI^kS^dD^d) mice were obtained from Jackson

Laboratories, Bar Harbor, Me., or from the Frederick Cancer Research Facility of the National Cancer Institute.

Bone marrow transplantation: Bone marrow transplantation (BMT) was performed as previously described (Ildstad, S.T., S.M. Wren, J.A. Bluestone, S.A. Barbieri, and D.H. Sachs. 1985. Characterization of mixed allogeneic chimeras. Immunocompetence, in vitro reactivity, and genetic specificity of tolerance. J. Ex . Med. 162:231) . Briefly, recipient B10 mice, aged 12 to 16 weeks, were lethally irradiated (1025R, ¹³⁷Cs source, HOR/min) and reconstituted within 8 to 12

^' hours with BMC obtained from the tibiae and femora of sex-matched donors aged 6-14 weeks. Animals were housed in sterilized microisolator cages, in which they received autoclaved food and autoclaved acidified drinking water. Syngeneic (BIO) bone marrow was TCD using rabbit anti-mouse brain (RAMB) serum, as previously described (Ildstad, S.T., et al., 1985, supra) . Ten to fifteen million untreated fully MHC- mismatched allogeneic spleen cells, varying in dose from 6.5xl0⁶ (A/J spleen cells) to 35xl0⁶ (B10.D2 spleen cells) . All BMC and spleen cells were co-administered in a single 1 ml intravenous injection. Irradiation controls received no BMC or spleen cells and died 7 to 12 days after irradiation. In order to avoid any cage-related effects on experimental results, animals were randomized both before assigning the experimental groups, and after BMT, so that animals from different experimental groups were randomly mixed in each cage. Survival was checked on a daily basis for 100 days.

Leukemia model: 5x10⁶ syngeneic (BIO) BMC, TCD as previously described (S.T. Ildstad, et al., 1985, supra) , 10 to 15 million untreated A/J BMC (except where indicated) , 6- a subline of the B6 T cell leukemia/lymphoma EL4, were thawed from frozen vials and maintained in culture for 4 to 14 days prior to each experiment. BMC, EL4 cells, and spleen calls were co-administered in a single 1 ml intravenous injection, as described (M. Sykes, Z. Bukhari, D.H. Sachs, 1989, Bone Marrow Transplant 4.:465). Animals were randomized as described (M. Sykes, Z. Bukhari, D.H. Sachs, 1989, Bone Marrow Transplant 4.:465), and survival was checked

SUBSTITUTE on a daily basis for 100 days.

IL-2 administration: The indicated doses of recombinant human IL-2, provided by Cetus corporation (Emeryville, CA) , were injected intraperitoneally in 0.2 ml of Hanks Balanced Salt Solution. Unless otherwise indicated, the first dose of IL-2 was administered one to three hours before BMT, and approximately every 12 hours thereafter for a total of 10 doses. As a control for IL-2 toxicity, additional irradiated animals received IL-2 plus TCD syngeneic marrow with or without allogeneic marrow, and without allogeneic spleen cells.

Monoclonal antibodies (Abs) : FITC-conjugated mAb (anti-K^b) (Sherman, L.A. , and CP. Randolph. 1981. Monoclonal anti-H-2Kb antibodies detect serological differences between H-2kb mutants. Immunoqenetics .12.:183) and biotinylated mAb 34-2-12 (anti-D^d) (Ozato, K. , N.M. Mayer, and D.H. Sachs. 1982. Monoclonal antibodies to mouse major histocompatibility complex antigens. Transplantation. 34.:113) were prepared by standard methods using antibodies purified from ascites using Protein A-Sepharose 4B beads (Pharmacia, Uppsala, Sweden) .

Phenotyping of chimeras: Phenotyping was performed 9 to 15 weeks after BMT. Animals were bled and peripheral blood mononuclear cells (PBMC) were isolated as described (Ildstad, S.T., et al., 1985, supra) . PBMC from each animal were then split into two tubes, and staining with mAbs were performed as described (Sykes, M. , M. Sheard, and D.H. Sachs. 1988. Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD. . Immunol. 141.:2282). Staining with both donor-specific and host-specific mAbs was performed on each chimera and control animal. One-color flow cytometry (FCM) was performed as described (Segal, D.M., S.O. Sharrow, J.F. Jones,^' and R.P. Sirigaanian. 1981. Fc(IgG) receptors on rat basophilic leukemia cells. J. Immunol. 126:138) . In all experiments, percent staining was determined from one-color fluorescence histograms and comparison with those obtained from normal donor and host-type animals, which were used as positive and negative controls. The percentage of cells considered positive after staining with a mAb was determined using a cutoff for positivity chosen as the fluorescence level at the beginning of the positive peak of the positive control strain. The relative percent staining of a chimera with a mAb was calculated using the formula:

(Chimera _Cpositive)-(Negative control ^positive) x 100%

(Positive control 5_positive)-(Negative control ^positive)

Since the mAbs used were allele-specific for class I H- 2 antigens,nearly 100% of cells from positive control animals, and 0% of cells from negative control animals, stained with each mAb in every experiment.

Statistical analysis: Survival probability was determined using the censored data technique of Kaplan-Meier, and statistical significance was determined using the method of Wilcoxon and Breslow. Since this method of analysis attributes increased weight to the early portion of a survival curve, a two- tailed stratified log rank survival test was substituted when the question of protection from late

SUBSTITUTE mortality was specifically being addressed. All statistical results are expressed as P values, and values less than 0.05 are considered to be significant.

Example l IL-2 +/- TCD syngeneic BMT prevents moderate GVHD

The results in Figure 1 show the effects of IL-2 and TCD syngeneic marrow on mortality from a moderately severe GVHD, which caused early mortality in one third of control animals. The survival of lethally irradiated BIO control mice given 15xl0⁶ A/J BMC plus 8xl0⁶ A/J spleen cells is shown by the solid line in Figure 1, panels B to D. All animals presumably succumbed to GVHD, since control animals not receiving A/J spleen cells demonstrated excellent survival (Figure 1A) . The results in Figure IB demonstrate that, while TCD syngeneic marrow prevented early GVHD mortality, all animals eventually succumbed to chronic GVHD, and the overall survival curve was not significantly different from that of the controls. However, similar to previous reports (Sprent, et al., 1988, supra), GVHD mortality occurred in two phases, including an acute phase in which deaths occurred between days 7 and 15, and a more chronic phase, usually beginning after day 30. Therefore, separate statistical analyses were performed on the two phases of the survival curves. Analysis of the early portion of the curves (i.e., the first 25 days) revealed a statistically significant (P<0.02) protective effect of TCD syngeneic marrow against mortality. When the results of this experiment were combined with three others involving similarly mild early GVHD mortality, a significant protective effect of TCD syngeneic marrow was again demonstrated (data not shown) .

The effect of IL-2 administration on GVHD mortality is shown in Figure 1C. In these animals, IL- 2 was found to protect against both early and late GVHD mortality (Figure 1C; P<0.008).

Figure ID shows the effect of combined treatment with TCD syngeneic marrow plus IL-2, 50,000 units (U) twice daily from days 0 to 4, on GVHD mortality. This combined regimen significantly reduced both early and late GVHD mortality, so that 63% of animals survived greater than 100 days, compared with only 7% survival among animals receiving neither IL-2 nor TCD syngeneic marrow (P<0.0006). Similar protection from late GVHD mortality by this treatment regimen has been reproducibly observed in another strain combination, B10.D2 into B10 (P<0.003 for the combined results of three experiments; N-27 in each group) . Although the difference in survival between the groups receiving IL-2 with or without TCD syngeneic marrow did not achieve statistical significance, long- term survival was slightly greater in recipients of TCD syngeneic marrow (63% versus 46%) , possibly reflecting the improved protection from acute GVHD seen in the group receiving TCD marrow (see below) . In two additional experiments comparing chronic GVHD mortality in animals receiving IL-2 with or without TCD syngeneic marrow, the late mortality curves of both groups were also similar (see below) .

Example 2 IL-2 +/- TCD syngeneic BMT prevents acute GVHD

The effects of IL-2 on acute mortality due to a more potent GVHD were examined next. In most experiments, administration of greater than 8xl0⁶ A/J spleen cells was sufficient to kill control recipients before day 15, as is shown by the solid line in Figure 2 , panels B to D, for animals receiving llxl0₆ A/J BMC plus 9xl0⁶ A/J spleen cells. Since animals receiving BMC without spleen cells demonstrated excellent ^■ survival (Figure 2A) , mortality was most likely due to GVHD. As shown in Figure 2B, administration of TCD syngeneic marrow without IL-2 had no effect on the rapid mortality from this A/J lymphocyte inoculum (P<0.05), and similar results have been observed in most experiments in which the majority of control animals died in the acute phase of GVHD. The effects of IL-2 on such mortality in mice receiving A/J cells with or without TCD syngeneic marrow were examined.

The results in Figure 2C show that IL-2 (10,000 U twice daily for 5 days) provided no protection against acute GVHD mortality when given without TCD syngeneic marrow (P<0.05). In contrast, administration of TCD syngeneic marrow plus IL-2 was associated with significant protection from GVHD mortality (Figure 2D; P<0.02). Thus, co-administration of TCD syngeneic marrow and IL- 2 was necessary to protect against mortality from the potent GVHD observed in this experiment. In an additional experiment in which IL-2 alone did not provide optimal protection, a similar effect of TCD syngeneic marrow was observed (data not shown) . In other experiments, IL-2 alone was capable of producing marked protection against acute GVHD mortality (e.g., Figure 3) . Nevertheless, maximal early survival was achieved in recipients of TCD syngeneic marrow along with IL-2 in 5 of 5 experiments (e.g. Figures 2 and 3). Example 3 Relationship of IL-2 dose to prevention of GVHD

The dose-response relationship of IL-2 and protection from GVHD mortality was examined next. In four of five experiments the degree of protection from acute GVHD mortality was directly proportional to^' the dose of IL-2 administered. These results are summarized in Table 1, below; the difference in mortality in recipients of the 10,000 versus the 50,000 U dose was significant, but only reflected differences in acute GVHD mortality (see 25 day survival, Table 1) . Only a small difference in long-term survival was apparent between the two groups, suggesting an increase in chronic GVHD mortality in recipients of the higher compared with the lower IL-2 dose (100 day survival,

Table 1) . The magnitude of the acute protective effect in a fifth experiment was inversely proportional to the dose (10,000, 25,000 or 50,000 U twice daily for five days) of IL-2 administered. This result was considered to be due to aberrant IL-2 toxicity (discussed above) and was not included in the summary presented in Table 1. Control animals receiving TCD syngeneic marrow and/or A/J marrow with or without IL-2 demonstrated uniformly excellent survival (data not shown) . TABLE 1

Dose-Response Relation of IL-2 to GVHD Mortality B10(-T)+ A/J BM + A/J Spleen —> BIO

Number of Survivors IL-2 Dose" (% Survival) MST^b (days) P^c Day 25 Day 100

O 6 (16%) 5 (13%) (N=38)

<0.002

10,000 U 21 (55%) 14 (37%) 47 (N=38) <0.03

50,000 U 35 (91%) 16 (46%) 89 (N=35)

" The indicated dose was administered twice daily for five days beginning immediately prior to bone marrow transplantation. ^b MST, median survival time determined from Kaplan-Meier plots. ^c P value comparing group above and below the indicated value. For group receiving 50,000 U IL-2, P<0.0001 compared with group not receiving IL-2. All P values were determined using the method of Wilcoxon and

Breslow.

SUBSTITUTE SHEET Example 4 Effect of timing of IL-2 on prevention of GVHD Several investigators have reported that in vivo administration of IL-2 is associated with acceleration of GVHD mortality (Sprent, et al., 1988, supra;18;19) . One possible explanation for this * discrepancy with the present results was that IL-2 was administered by those workers for a prolonged period, or was begun with a delay of 7 or 8 days after BMT (Sprent, et al., 1988, supra; Malkovsky, et al., 1986, supra) , whereas here administration of IL-2 was begun on the day of BMT and completed after less than 5 days. To assess this possibility, comparisons were made of survival in lethally irradiated BIO recipients of TCD BIO marrow plus A/J BMC and spleen cells without IL-2, or with 10,000 U IL-2 administered twice daily for 5 days beginning either on the day of irradiation and BMT, or 7 days later. The results, shown in Figure 4, indicate that IL-2 was protective only when administration was begun on the day of BMT (P<0.01). Administration of IL-2 beginning on day 7 was associated with a significant acceleration of GVHD mortality (P<0.0005), consistent with previous reports (Sprent, et al., 1988, supra; Malkovsky, et al., 1986, supra) . Similar results were obtained in a repeat experiment utilizing the higher dose of IL-2 (50,000 U).

In other experiments, lethally irradiated B10 recipients of TCD B10 marrow plus A/J BMC and spleen cells were treated with two courses of IL-2 administered twice daily for 5 days, with the first course beginning on the day of irradiation and BMT, and the second 7 days later (data not shown) . The protective effect of the initial IL-2 course against GVHD was not obviated by the later course.

Although the first dose of IL-2 was administered immediately before BMT in all experiments reported here, such timing was not critical, since additional experiments demonstrated that delaying administration until one hour after BMT did not reduce the anti-GVHD effect of IL-2 (data not shown) . Administration of a single high dose (50,000 U) of IL-2 (immediately preceding BMT) did not protect against acute GVHD mortality (Figure 5) .

Since animals in some experiments began to appear ill on the fourth day of high dose (50,000 U) IL-2 administration, showing lethargy, hunching, and ruffled fur, it seemed possible that this dose of IL-2 might be producing cumulative toxicity. Therefore, the effect of a shortened 2.5-day (5 dose) course of high- dose IL-2 on GVHD mortality was examined. As shown in Figure 5, this shortened course was at least as protective against early GVHD mortality as was a full 5-day course, and the animals showed no clinical evidence for adverse effects. Similar results were obtained in a repeat experiment. Also, no differences in later mortality have been seen after 100 days of follow-up.

Example 5 Effect of IL-2 on engraftment To examine the effects of IL-2 on alloengraftment, the PBL of long-term BMT survivors were phenotyped using mAbs and FCM. No differences were observed in the level of allogeneic reconstitution between animals receiving or not receiving IL-2 (10,000 to 50,000 U twice daily for 5 days for one or two courses) along with allogeneic (A/J or B10.D2) spleen cells, BMC, and TCD syngeneic marrow. Examples of FCM profiles from such animals are shown in Figure 6. Most animals in all groups, regardless of whether or riot spleen cells were administered, demonstrated complete allogeneic lymphopoietic repopulation, similar to the results shown in Figure 6. In some animals receiving IL-2 plus allogeneic BMC and TCD syngeneic BMC without allogeneic spleen cells, however, a small, negative peak representing about one to ten percent of cells was evident on staining with antibody recognizing donor H-2 antigens. This negative peak corresponded to a positive peak on staining with antibody recognizing host H-2 antigens, and tended to disappear with time; such host-type cells were detected in 6 of 7 recipients tested before day 70, and in only 5 of 27 such animals tested after day 95. One of thirteen simultaneous control recipients of TCD syngeneic marrow and allogeneic marrow without IL-2 demonstrated such a peak. Of 21 animals receiving allogeneic spleen cells in addition to TCD syngeneic marrow, allogeneic marrow and IL-2, none showed any evidence for repopulation by host-type cells at any time.

Example 6 IL-2 permits an anti-leukemic effect of BMT

The_rEL4 H-2^b leukemia model in mixed allogeneic BMT recipients, which was recently described by the present inventors (M. Sykes, Z. Bukhari, D.H. Sachs, 1989, Bone Marrow Transplant. 4.:465) , was used for the present tests. Female C57BL/10nCR (B10) H-2^b mice were lethally irradiated and reconstituted with 5xl0⁶ T cell- depleted (TCD) syngeneic bone marrow cells (BMC) plus 5xl0² EL4 leukemia cells (M. Sykes, Z. Bukhari, D.H. Sachs, 1989, Bone Marrow Transplant. 4:465). All such recipients died of tumor by day 19 (Figure 7A) .

Treatment with IL-2 on days 0 through 3 led to slight prolongation of survival, with all animals dying by day 23 (Figure 7A; P<0.02). The addition of 10⁷ A/J bone marrow cells (BMC) plus 10⁷ A/J spleen cells to inocula containing TCD syngeneic marrow plus EL4 was associated with a highly significant anti-leukemic effect in animals also receiving IL-2 (Figure 7A) (P<0.002). The median survival time was extended to 33 days, and 2 of 10 animals survived greater than 100 days. An anti- leukemic effect was also observed in animals receiving a similar regimen without TCD syngeneic marrow, but a greater percentage of these animals died in the second week from GVHD, rendering the improvement in survival not statistically significant (P= 0.09). Figure IB confirms, in the same experiment, that IL-2 was necessary in order to prevent death from GVHD. Fifty percent of control animals receiving A/J bone marrow plus spleen cells without EL4 or IL-2 died by day 10 from acute GVHD, and the remainder died of chronic GVND, which, in our model, begins to produce mortality at approximately 25 to 40 days. In contrast, 70% of control animals receiving IL-2 in addition to a similar A/J inoculum survived longer than 100 days, confirming the above noted protective effect of IL-2 against both acute and chronic GVHD (P<0.01). The most significant protection from acute GVHD was observed in animals receiving TCD syngeneic marrow along with the same A/J

SUBSTITUTE SHEET inoculum plus IL-2 (P<0.001), consistent with previous results. There were no acute GVHD deaths in this group, and 80% of the animals survived long-term (Figure 7B) . Control animals receiving TCD syngeneic plus A/J BMC without spleen cells with or without IL-2 demonstrated excellent survival (100% at 100 days; not shown) ; thus, mortality among the groups shown in Figure 7B reflected GVHD caused by administration of allogeneic spleen cells. The strong anti-leukemic effect observed in

Figure 7 required the co-administration of A/J spleen cells, since, as shown in Figure 8A, A/J BMC plus TCD syngeneic marrow administered without A/J spleen cells produced a minimal prolongation (P<0.05) of survival (median survival time [MST] 24 days) compared to recipients of TCD syngeneic marrow, EL4 and IL-2 (MST 22 days) . In contrast, animals receiving 6xl0⁶ A/J spleen cells in addition to EL4, A/J BMC, TCD syngeneic marrow and IL-2, demonstrated markedly improved survival (MST 39 days: P<0.0001), with 3 of 10 animals surviving longer than 50 days (Figure 8A) . Figure 8B demonstrates in this experiment that IL-2 was necessary to prevent acute GVHD mortality, since most control recipients of EL4 plus A/J BMC and spleen cells without IL-2 were dead by day 12, before leukemic deaths even began in recipients σf syngeneic marrow, EL4, and IL-2. Recipients of similar A/J inocula without EL4 cells showed an almost identical mortality pattern, indicating that EL4 cells had no effect on GVHD. ^"The addition of IL-2 to this regimen was associated with significant protection from GVHD (P<0.01), and a highly significant anti-leukemic effect could be detected; recipients of the same treatment plus TCD syngeneic marrow again enjoyed optimal survival (Figure 8B) (P<0.002). In this experiment, TCD syngeneic marrow administered with A/J BMC and spleen cells in the absence of IL-2 did not protect against GVHD mortality (Figure 8B) , confirming the lack of efficacy of TCD syngeneic marrow alone against very severe GVHD noted above. Control animals receiving A/J BMC plus TCD syngeneic marrow with or without IL-2 demonstrated 100% survival (not shown) .

Example 7 IL-2 does not reduce anti-leukemic effect of BMT

Since animals receiving A/J spleen cells without IL-2 died of GVHD, it could not be determined from the above studies whether or not the magnitude of the anti-leukemic effect of A/J spleen cells was reduced by the co-administration of IL-2. In order to address this question, we examined the effect of IL-2 administration on the anti-leukemic effect of a larger dose of A/J BMC (3xl0⁷) , which, due to its low T cell content, does not cause acute GVHD (M. Sykes, Z. Bukhari, D.H. Sachs, 1989, Bone Marrow Transplant 4.:465). The lack of GVHD mortality is confirmed in Figure 9, in which control recipients of A/J BMC plus TCD BIO BMC with or without IL-2 demonstrated excellent survival. The results presented in Figure 9 demonstrate that this dose of A/J BMC, when co- administered with TCD syngeneic marrow, mediated a small, but significant anti-leukemic effect (P<0.004), similar to previous results (M. Sykes, D.H. Sachs,

1989, Transplant. Proc. .21:3022). The addition of IL-2 to the regimen did not reduce the magnitude of this anti-leukemic effect (P<0.004; Figure 9). In the same experiment, administration of IL-2 to recipients of TCD syngeneic marrow, A/J BMC and A/J spleen cells was associated with an increase in survival from 22% in control animals not receiving IL-2 to 68% in recipients of IL-2 (data not shown) . Thus, IL-2 can protect^" against GVHD mortality without reducing the magnitude of even a weak anti-leukemic effect.

* * * For purposes of completing the background description and present disclosure, each of the published articles, patents and patent applications heretofore identified in this specification are hereby incorporated by reference into the specification. The foregoing invention has been described in some detail for purposes of clarity and understanding. It will also be obvious that various changes and combinations in form and detail can be made without departing from the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method for facilitating engraftment of allogeneic bone marrow in a mammal comprising administration of interleukin 2 to said mammal beginning about the time of marrow transplantation, wherein the dose rate and duration of said administration is effective to provide protection against graft-versus-host disease.

2. The method of claim 1 wherein said interleukin 2 is administered in the form of multiple discrete uniform doses at regular intervals.

3. The method of claim 2 wherein the first of said doses is administered within the period from about three hours before said transplantation to about one hour after said transplantation and subsequent doses are administered approximately every twelve hours thereafter.

4. The method of claim 3 wherein the total number of doses of interleukin 2 is greater than one but not more than ten doses.

5. The method of claim 1 further comprising transplanting T cell depleted syngeneic bone marrow to said mammal prior to or during transplantation of said allogeneic bone marrow.

6. The method of claim 1 or of claim 5 further comprising transplanting allogeneic ^" " lymphopoietic cells in addition to said allogeneic bone marrow.

7. A method for facilitating engraftment of an allogeneic organ or tissue other than bone marrow in a mammal comprising the steps of: i. engraftment into said mammal of allogeneic bone marrow cells from the proposed organ or tissue donor; ii. optionally, engraftment of additional allogeneic lymphopoietic cells from said donor; iii. administration of interleukin 2 to said mammal beginning about the time of marrow transplantation, wherein the dose rate and duration of said administration is effective to provide protection against graft-versus-host disease, whereby said mammal becomes tolerant to subsequent organ or tissue transplants from said donor.