Immunotherapy in high-risk chemotherapy-resistant patients with metastatic solid tumors and hematological malignancies using intentionally mismatched donor lymphocytes activated with rIL-2: a phase I study.

The feasibility and safety of immunotherapy mediated by intentionally mismatched rIL-2 activated killer lymphocytes (IMAK) with no prior stem cell engraftment was investigated in patients with advanced chemotherapy-resistant hematological malignancies and metastatic solid tumors. Our goals were to maximize anti-cancer activity by using intentionally mismatched donor lymphocytes; amplify killing of target cancer cells by rIL-2 activation of killer cells in vitro and in vivo, and avoid the risk of graft-versus-host disease (GVHD) by anticipated rejection of alloreactive donor lymphocytes. Conditioning consisted of 5 days of fludarabine 25 mg/m(2) or a single dose of cyclophosphamide 1,000 mg/m(2), 2 subcutaneous injections of alpha interferon (IFN) 3 x 10(6) and COX2 inhibitors, followed by administration of IMAK (65 +/- 5 CD3(+)CD56(-); 17 +/- 5 CD3(-)CD56(+)) in conjunction with low dose subcutaneous rIL-2 (6 x 10(6) IU/m(2)/day) for 5 days for continuous activation of alloreactive donor lymphocytes prior to their anticipated rejection. Here, we present our phase 1 clinical study data in a cohort of 40 high-risk patients with metastatic solid tumors and hematological malignancies. Treatment was accompanied by some malaise and occasional self-limited fever but otherwise well tolerated on an outpatient basis. Transient engraftment of donor cells was documented in two patients and only one developed self-limited grade 1 GVHD. Among patients with chemotherapy-resistant disease, long-term progression-free survival was recorded in 5 of 21 evaluable patients with metastatic solid tumors and in four of five patients with hematological malignancies. We conclude that the proposed procedure is feasible, safe, and potentially effective, with some otherwise resistant cancer patients long-term disease-free, thus justifying larger Phase II studies in patients with hematological malignancies and metastatic solid tumors, preferably at a stage of minimal residual disease with the goal in mind to eradicate all malignant cells at an early stage of the disease.

Induction of graft-versus-leukemia (GVL) effect without graft-versus-host disease (GVHD) by pretransplant donor treatment with immunomodulators.

Pretransplant donor treatment with immunomodulators such as complete Freund's adjuvant (CFA) or oligodeoxynucleotide sequences expressing CpG motifs (CpG), was applied in sublethally irradiated host mice inoculated with murine models of mammary carcinoma (4T1) or B cell leukemia (BCL1). Spleen cells or IL-2 activated splenocytes (lymphokine activated killer [LAK]) derived from donor mice treated with CpG emulsified in incomplete Freund's adjuvant (IFA), (CpG + IFA) did not cause graft-versus-host disease (GVHD), but were not efficient enough to induce a significant graft-versus-tumor (GVT) response against 4T1 cells. In contrast, an efficient graft-versus-leukemia (GVL) effect was evident in BCL1-bearing mice inoculated with spleen cells from donors pretreated with CFA or CpG + IFA. Pretransplant donor treatment with CFA prolonged survival to a median of 62 days with 3 of 27 mice remaining GVHD- and leukemia-free for >200 days, compared to GVHD-related death of all mice inoculated with naïve cells (median 17 days), or leukemia-related death of all mice inoculated with leukemia cells (median of 27 days). Pretransplant donor treatment with CpG + IFA exerted a more efficient GVL effect with reduced GVHD resulting in 12 of 26 GVHD- and leukemia-free survivors for >200 days. Our results suggest that it may be possible to prevent GVHD while sparing an efficient GVL effect by using pretransplant donor treatment with immunomodulators prior to allogeneic stem cell transplantation and/or donor lymphocyte infusions in hematologic malignancies.

Immunoregulation of GVHD by triggering the innate immune system with CpG.

Stimulation of Toll-like receptors by oligodeoxynucleotide sequences containing a CpG motif provides signals capable of triggering the innate and adaptive immune systems, thereby leading either to stimulation or suppression of immunoreactivities. Similar immunoregulatory capabilities are necessary for achieving the fine balance between engraftment and graft-versus-host disease required in the setup of allogeneic cell therapy. Ligation of CpG to its Toll-like receptors can be accomplished by treatment of the host or pretransplant treatment of the donor in vivo. These different strategies are presented in this review, which summarizes the attempts to maximize beneficial alloreactivity against malignant or other undesirable host cells, while controlling graft-versus-host disease.

CpG-induced myeloid CD11b+Gr-1+ cells efficiently suppress T cell-mediated immunoreactivity and graft-versus-host disease in a murine model of allogeneic cell therapy.

Transplantation of mismatched allografts in irradiated recipients results in lethal graft- versus-host disease (GVHD). In our study, pretransplantation donor treatment with CpG, administered either alone or emulsified in incomplete Freund's adjuvant, efficiently prevented GVHD in sublethally irradiated recipients of haploidentical (H-2(b) into H-2(b/d)) and fully mismatched (H-2(b) into H-2(d)) allografts. CpG treatment of donor mice caused an accumulation of double-positive CD11bGr-1 cells in their blood and spleens, whereas treatment with CpG+IFA resulted in an even greater accumulation of these cells. Isolated CD11b(+) cells from the spleens of CpG+IFA-treated mice efficiently suppressed alloreactivity in vitro (> 92%), as determined by co-culturing these cells in mixed lymphocyte reactions. After CpG+IFA treatment, a T cell-depleted fraction enriched with CD11b(+)Gr-1(+) cells, acting as myeloid suppressor cells, was able to efficiently prevent GVHD induced by naïve T cells in the sublethally irradiated recipients: 20/21 mice remained GVHD-free survivors for more than 200 days. Splenocytes from CpG+IFA-treated mice displayed enhanced interleukin (IL)-6, IL-10, and interferon-gamma production, reduced T cell allogeneic and mitogenic responses, as well as failure of T cells to induce GVHD. In summary, CpG treatment led to impaired T cell function, enriched myeloid suppressor cells and regulatory cytokine production, which together appear to suppress alloreactivity and protect against the development of GVHD. We hypothesize that similar immunoregulatory effects could be applied experimentally in a clinical setting when inhibition of alloreactivity is required in recipients of stem cell allografts.

Mesenchymal stromal cells lose their immunosuppressive potential after allotransplantation.

OBJECTIVE: The stem cell fraction of mesenchymal stromal cells (MSCs) is capable of self-renewal and under inductive conditions differentiates into bone, cartilage, hematopoietic stroma, and other mesenchymal tissues. Therefore, MSCs represent a promising source for hard tissue repair therapies. MSCs are also immunosuppressive and prevent activation of allogeneic lymphocytes in vitro. Thus it has been suggested that they might be able to engraft in allogeneic recipients and downregulate recipients' immunity. In this study we examined whether MSCs retain their immunomodulating properties in vivo after allotransplantation. MATERIALS AND METHODS: MSCs were propagated from bone marrow (BM), placenta, or umbilical cord tissues. Using a murine parental-into-F1 model of graft-vs-host disease (GVHD) we tried to control GVHD by intravenous transplanting parental or recipient MSCs together with parental lymphocytes (day 0) and on days +7 and +14. MSCs' immunosuppressive potential in vivo was also examined by comparing their ability to construct ectopic bone after local transplantation with osteogenic inductor (demineralized bone matrix) under the kidney capsule of syngeneic and allogeneic recipients. RESULTS: Repeated IV MSC injections failed to reduce GVHD-related recipient mortality. Local implantation of MSCs propagated from BM, placenta or umbilical cord resulted in ectopic bone formation in syngeneic recipients and in transplant rejection by allogeneic mice. CONCLUSION: MSCs lose their immunosuppressive potential in mismatched setting.

Induction of long-lasting antitumor immunity by concomitant cell therapy with allogeneic lymphocytes and trifunctional bispecific antibody.

OBJECTIVE: Use of a trifunctional bispecific antibody (trAb) given concomitantly with allogeneic cell therapy to achieve an anti tumor effect without graft-vs-host disease (GVHD). MATERIALS AND METHODS: A trAb-directed against murine CD3 and human epithelial cell adhesion molecule (EpCAM) (BiLu), was given alone or concomitantly with interleukin (IL)-2-activated (LAK) H-2(b) donor splenocytes to H-2(d/b) mice inoculated with murine melanoma cells transfected with human EpCAM. RESULTS: A total of 32/38 mice treated with BiLu and LAK splenocytes, were tumor-free survivors without GVHD for >200 days following inoculation of a 100% lethal tumor dose (5 x 10(4)). Of 28 disease-free surviving mice previously treated with LAK splenocytes and BiLu, 24 mice proved resistance to a second tumor challenge of 10(4) cells given >210 days following the first tumor inoculation with no evidence of disease for >150 days. In contrast, only 4 of 13 disease-free survivor mice treated with naïve splenocytes and BiLu, and 5 of 10 disease-free survivor controls treated with BiLu only, resisted the second tumor challenge. Induction of antitumor immunity was more efficient and long-lasting (>150 days) in mice previously injected with a lethal tumor cell dose of 5 x 10(4) cells than in mice previously inoculated with 5 x 10(3) tumor cells. CONCLUSION: Concomitantly treatment of allogeneic LAK cells and trAb-induced an efficient long-lasting antitumor immunity. Considering the documented efficacy of anti-EpCAM bispecific antibody in various metastatic cancers, clinical application of our approach may be justified in patients with minimal residual disease at high risk for tumor recurrence.

Pretransplant treatment of donors with immunomodulators to control graft-versus-host disease (GVHD) in transplant recipients.

OBJECTIVE: Prevention of graft-versus-host disease (GVHD) by pretransplant donor treatment with known immunomodulators like complete Freund's adjuvant (CFA) and synthetic oligo-deoxynucleotides expressing CpG motifs (CpG). METHODS: Induction of GVHD by inoculation of C57BL/6 (C57) splenocytes into sublethally irradiated (BALB/c x C57BL/6) F1 (F1) mice. Splenocytes were derived from either naive C57 mice or from C57 mice that were treated previously with the immunomodulators. RESULTS: Inoculation of CFA or CpG into C57 mice led to an increase in the total number of spleen cells and resulted in activation of immunoregulatory cells that significantly suppressed mixed allogeneic lymphocyte reaction in vitro. CFA-treated C57 splenocytes led to GVHD-related death in only 14 out of 61 F1 recipients while the remaining 47 mice survived without disease for more than 200 days. Pretransplant treatment of donor C57 mice with GpG emulsified in incomplete Freund's adjuvant resulted in 19/20 GVHD-free survivors of sublethally irradiated F1 mice for more than 200 days. In contrast, naive C57 splenocytes injected into sublethally irradiated F1 recipients induced severe GVHD, which resulted in the death of 77/78 recipient mice (median of survival was 16 days). CONCLUSION: Our results suggest that adjuvant-induced immunoregulation of donor cells prior to allogeneic cell therapy may augur a new strategy that will bring the benefits of safe cellular immunotherapy aiming to eradicate malignant and nonmalignant pathological cells while avoiding or minimizing the risk of GVHD.

Use of trifunctional bispecific antibodies to prevent graft versus host disease induced by allogeneic lymphocytes.

A trifunctional bispecific antibody (BiLu) directed against murine CD3 and human epithelial-cell adhesion molecule (EpCAM) was tested for its ability to improve cell-mediated adoptive immunotherapy in a murine model of B16 melanoma cells transfected with human EpCAM. Intraperitoneal inoculation of naive C57BL/6 (C57) splenocytes induced lethal graft versus host disease (GVHD) in 85% to 97% of sublethally irradiated (BALB/c x C57BL/6) F1 (F1) hosts inoculated intraperitoneally with a sublethal or lethal dose of melanoma cells. BiLu antibodies given intraperitoneally concomitantly with alloreactive C57 cells effectively prevented GVHD-related and tumor-related death in 16 of 25 F1 mice inoculated with a sublethal tumor-cell dose and in 10 of 20 mice inoculated with a lethal tumor-cell dose over a follow-up period of more than 200 days. BiLu treatment also efficiently prevented severe GVHD, which was induced by high doses of BALB/c-derived splenocytes. Trifunctional bispecific antibodies (TbsAbs) capable of cross-linking T lymphocytes, natural killer, and other FcgammaR-positive effector cells, via their Fc region, to the tumor cells may be applied together with adoptive allogeneic-cell therapy to maximize antitumor responses while acting on GVHD in patients with minimal residual disease.

Depletion of alloantigen-primed lymphocytes overcomes resistance to allogeneic bone marrow in mildly conditioned recipients.

OBJECTIVE: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. MATERIALS AND METHODS: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. RESULTS: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. CONCLUSION: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy.

A novel approach for prevention of lethal GVHD by selective elimination of alloreactive donor lymphocytes prior to stem cell transplantation.

OBJECTIVE: Experiments were designed to investigate a new concept aiming for induction of graft-vs-malignancy (GVM) effect prior to stem cell transplantation (SCT). Mismatched lymphocytes given pre-SCT will be followed by a selective elimination of alloreactive donor lymphocytes, thus avoiding lethal graft-vs-host disease (GVHD). METHODS: Recipient mice treated with sublethal total-body irradiation (TBI) received a single injection of allogeneic splenocytes, either naïve or rIL-2 activated (ADL), for induction of GVHD. To prevent lethal GVHD, cyclophosphamide (Cy) (200 mg/kg) or TBI (9 Gy) were given 4 days after cell inoculation. One day later, treated mice were rescued with syngeneic bone marrow cells. RESULTS: Both Cy and TBI significantly (p < 0.001) prevented GVHD in all recipients inoculated with either naïve cells or ADL and all recipients survived more than 250 days. Control mice not rescued with CY or TBI died of GVHD within 20 to 25 days. A significant proportion of recipients inoculated with 4T1 tumor cells, treated with ADL followed by TBI 9 Gy, survived more than 250 days disease-free in comparison with 22 days in untreated control mice (p <0.001). CONCLUSIONS: Attempting to induce short, yet effective, GVM effects before rather than after SCT, thus avoiding lethal GVHD, may represent an innovative approach for more effective yet safer use of SCT for tumor immunotherapy.

Effect of KRN7000 on induced graft-vs-host disease.

OBJECTIVE: Graft-vs-host disease (GVHD) is a serious complication of allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), for which no effective therapy exists. In our study, KRN7000, a synthetic analog of alpha-galactosylceramide, known for its ability to activate natural killer T cells, was tested for its ability to prevent onset of GVHD in a murine model of haploidentical major histocompatible (MHC) mismatched hematopoietic cells. METHODS: Irradiated (BALB/cXC57BL/6)F1 mice were inoculated with parental C57BL/6 splenocytes with or without SCT. KRN7000 was given intraperitoneally as single or multiple doses at 100 microg/kg/dose and mice were followed up for GVHD clinical symptoms and for survival. The effect of KRN7000 treatment was also tested in vitro for the induction of suppression of alloreactivity in mixed lymphocyte reaction (MLR). RESULTS: KRN7000 prevented development of GVHD symptoms in almost all mice and 52/53 mice maintained a healthy profile for more than 235 days. Most vehicle-treated mice or untreated controls died of GVHD within a median of 3 weeks. KRN7000 treatment did not prevent engraftment of donor cells following sublethal total-body irradiation (TBI) and allowed durable persistence of donor cells following lethal TBI and SCT. Splenocytes derived from KRN7000-treated mice suppressed efficiently mixed lymphocyte reaction (MLR) in vitro. CONCLUSION: GVHD induced by alloreactive lymphocytes can be prevented by KRN7000. GVHD prevention may be accomplished by regulation of T cell function and might thus provide a new modality for safer SCT and DLI.

Nonmyeloablative stem cell transplantation: reduced-intensity conditioning for cancer immunotherapy--from bench to patient bedside.

Despite major progress in treating hematologic malignancies and, to a lesser extent, metastatic solid tumors, much work remains ahead. With the anticancer potential of immunotherapy not yet fully exploited, patients with leukemia, malignant lymphoma, and other hematologic malignancies for which high-dose chemoradiotherapy is frequently recommended in conjunction with stem cell transplantation (SCT) can now benefit from the advantages of immunotherapy mediated by cytokines or alloreactive donor lymphocytes, while minimizing procedure-related toxicity and mortality. The feasibility of applying allogeneic cell-mediated immunotherapy in conjunction with allogeneic SCT following reduced-intensity conditioning, with minimal toxicity and no serious transplant-related complications, makes it possible to undertake such procedures on an outpatient basis as well as to offer an option for cure to elderly individuals and patients with less than optimal performance status. Being well tolerated, reduced-intensity transplants also offer a chance for cure to patients with otherwise resistant leukemia and malignant lymphoma who have relapsed after autologous SCT. Thus, the traditional obstacle of very high transplant-related toxicity and mortality due to multiorgan failure from cumulative toxicity of multiple anticancer agents and radiation therapy is overcome. Although immunotherapy mediated by allogeneic lymphocytes can be most effective, the immune potential of donor lymphocytes should be maximized by nonspecific or specific activation in vitro or in vivo, or both, for more effective eradication of resistant tumor cells, including in patients with bulky disease. More important is the challenge to target donor lymphocytes to the tumor and minimize their capacity to induce responses against normal host tissues, which frequently results in severe acute and chronic graft-versus-host disease (GVHD). Alternatively, donor lymphocytes should be eliminated as soon as tumor eradication is completed, or as soon as severe GVHD becomes prohibitive. Based on available experience, clinical application of innovative therapy, especially at the stage of minimal residual disease (MRD), may open new horizons for the treatment of malignancies considered until recently to be incurable. The feasibility of controlling cancer by targeted chemotherapy, best illustrated by the phenomenal activity of imatinib in patients with chronic myelogenous leukemia and, more recently, in gastrointestinal stromal tumors (including in patients fully resistant to all known anticancer agents) suggests that in the future, tumor-specific chemotherapy may represent the ultimate goal for achieving a stage of MRD with minimal multiorgan toxicity. Together, the combination of immunotherapy and targeted chemotherapy may provide the most logical approach for making real progress in controlling resistant hematologic malignancies and metastatic solid tumors.

Allogeneic versus syngeneic killer splenocytes as effector cells for the induction of graft-versus-tumor effect.

The effect of allogeneic versus syngeneic killer cells derived from normal or severe combined immunodeficiency disease (SCID) mice was evaluated for induction of antitumor reaction in a murine model of mammary carcinoma. Tumor cells of H-2d origin were injected intravenously into H-2(d/b) mice 24 hours after total body irradiation (4 Gy). On the following day, lymphokine-activated killer (LAK) splenocytes, derived from either minor (H-2d) or major (H-2b) histocompatibility complex (MHC)-mismatched parental normal mice or MHC (H-2b)-mismatched SCID mice, were given intravenously. LAK cells of H-2d normal or SCID mice, syngeneic to the tumor, were inoculated in parallel. The results show that LAK cells derived from minor histocompatibility complex-mismatched or MHC-mismatched parental normal mice improved the probability of tumor-free survival as compared with LAK cells syngeneic to the tumor cells, but they aggravated the severity of graft-versus-host disease. SCID splenocytes serving as a source of natural killer (NK) cells were expanded and activated in vitro by rIL-2 to obtain a sufficient number of DX5+ CD3- CD8- NK cells (SCID-LAK). H-2b SCID-LAK cells did not cause graft-versus-host disease and significantly delayed tumor growth compared with syngeneic H-2d SCID-LAK cells, as indicated by tumor colony assays in vitro and adoptive transfer experiments. However, the graft-versus-tumor effect was not long lasting, and treated mice finally died of tumor. Our results show an advantage of allogeneic over syngeneic cell therapy for achieving a graft-versus-tumor effect by rIL-2-activated T cells and NK cells. Periodic repetition of NK treatments may be required to achieve more durable antitumor effects.

Immunotherapy of hematologic malignancies and metastatic solid tumors in experimental animals and man.

New approaches are needed for maximizing specific responses against tumor cells resistant to chemotherapy. While cytokine therapy may amplify natural resistance against minimal residual disease, more robust anti-leukemia reactivity can be provided by allogeneic bone marrow transplantation (BMT) in conjunction with myeloablative, hence hazardous, conditioning, at the cost of graft-versus-host disease (GVHD). Documentation of the capacity of donor lymphocyte infusion (DLI) given late post BMT, when patients were off immunosuppression, in early 1987, with successful reversal of relapse and cure of patients fully resistant to maximally tolerated doses of chemoradiotherapy, with many patients alive and well >10-15 years later, indicated two important facts. First, resistant tumors are unlikely to be cured with higher doses of chemoradiotherapy that may harm the patient but not eliminate all his clonogenic tumor cells. Second, that under condition of tolerance to donor alloantigens, DLI may provide a cure to otherwise resistant patients. These observations paved the road for clinical application of non-myeloablative stem cell transplantation (NST), in the early 90s, based on a two-step procedure, first involving induction of transplantation tolerance to donor alloantigens by engraftment of donor stem cells, following safe lymphoablative rather than myeloablative conditioning. Second, use of donor lymphocytes for elimination of residual tumor or otherwise abnormal hematopoietic cells by immune-mediated graft-versus-host effects inducible by mobilized blood stem cell allografts containing larger inocula of donor T cells, or supported by post-grafting DLI when patients were off immunosuppressive modalities.

CD40 ligand-specific antibodies synergize with cyclophosphamide to promote long-term transplantation tolerance across MHC barriers but inhibit graft-vs-leukemia effects of transplanted cells.

OBJECTIVES: We previously demonstrated that allogeneic bone marrow transplantation (BMT) after low-dose total lymphoid irradiation (200 cGy) and depletion of donor-reactive cells with cyclophosphamide (Cy) converted recipients to graft-vs-host disease (GVHD)-free chimeras tolerant to donor skin grafts. BMT also generated strong graft-vs-leukemia (GVL) response. However, clinical application of the protocol was hampered by the requirement for a relatively high dose of Cy (200 mg/kg). In this study we have tried to minimize the Cy dose by a concomitant blockade of CD40-CD40L interaction. MATERIALS AND METHODS: Mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (BM(1)) and skin graft on day 0, injected with Cy (200 mg/kg or less) on day 1, and transplanted with a second C57BL/6 BM cell inoculum (BM(2)) on day 2. CD40L-specific antibody (MR1) was given with BM(1), BM(2), and 2 days later. Treated animals were monitored for survival, chimerism, and skin allograft rejection. The GVL potential of transplanted cells was examined in mice inoculated with BCL1 leukemia cells before irradiation. RESULTS: Blocking CD40-CD40L interaction with MR1 mAb allowed the reduction of a tolerance-generating Cy dose by 50%. Unfortunately, adding MR1 to the protocol reduced the GVL potential of the transplanted cells. Neither low-dose Cy nor antibodies alone could downregulate donor or recipient immune response. CONCLUSIONS: CD40L-specific antibodies synergize with Cy to induce bilateral transplantation tolerance. Therefore, their use may be beneficial for safer allogeneic BMT for nonmalignant indications. However, due to MR1-associated reduction of GVL effects, MR1 should be considered with caution as conditioning for BMT for leukemia-bearing recipients.

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

Donor lymphocyte infusion: the use of alloreactive and tumor-reactive lymphocytes for immunotherapy of malignant and nonmalignant diseases in conjunction with allogeneic stem cell transplantation.

Donor lymphocyte infusion (DLI), pioneered in Jerusalem in January 1987, represents the first proof of principle of the absolute efficacy of immunotherapy as a means of curing cancer. Immunotherapy with alloreactive donor lymphocytes can eliminate "the last tumor cell" even in patients with hematological malignancies resistant to maximally tolerated doses of chemoradiotherapy. Alloreactive lymphocytes that can mediate anti-tumor effects following induction of host-versus-graft tolerance induced by transplantation of donor stem cells, can induce graft-versus-malignancy (GVM) effects which are usually accompanied by graft-versus-host disease (GVHD). However, occasionally GVM effects may also be accomplished independently of clinically overt GVHD. Interestingly, allogeneic donor lymphocytes may also eliminate undesirable host-derived hematopoietic cells in a large number of nonmalignant indications including genetic diseases, diseases caused by deficiency of stem cell products, and autoimmune disorders mediated by self-reactive lymphocytes. The cumulative clinical experience suggests feasibility of effective induction of graft-versus-leukemia (GVL); graft-versus-lymphoma (GVLy); graft-versus-multiple myeloma, as well as graft-versus-solid tumors (GVT), well-documented in patients with renal and breast cancer, even in patients with resistant disease that have failed myeloablative chemoradiotherapy. These observations that suggested that cell therapy by donor lymphocytes is the main therapeutic benefit of bone marrow transplantation (BMT) led to development of the nonmyeloablative approach for safer allogeneic stem cell transplantation. Nonmyeloablative stem cell transplantation (NST) makes it possible to offer an option for cure to elderly patients with no upper age limit, as well as to patients with poor performance status not considered eligible for conventional BMT. Using well-tolerated NST regimen, allogeneic stem cell transplantation can be accomplished with minimal procedure-related toxicity and mortality, possibly even on an outpatient basis. Immunotherapy mediated by adoptive allogeneic cell-mediated immunotherapy can be further improved by utilizing specifically immune donor lymphocytes, thus maximizing their efficacy against undesirable target cells of host origin on the one hand, while minimizing their ontoward efficacy against normal cells of host origin that could result in GVHD on the other. Taken together, DLI and subsequently NST, may have opened new horizons for treatment of life-threatening malignant and nonmalignant disorders correctable by allogeneic stem cell transplantation. It is anticipated that further improvement of reactivity and specificity of donor lymphocytes will lead to safer clinical application of cell therapy for a larger number of indications toward improving disease-free survival in a large number of indications while minimizing immediate and late procedure-related complications.

Nonmyeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in preclinical models.

OBJECTIVE: We previously demonstrated that a combination of mild total lymphoid irradiation (TLI) with selective depletion of the host's donor-reactive cells allows for stable and graft-vs-host disease (GVHD)-free engraftment of allogeneic bone marrow (BM). In this study, we investigated the efficacy of this nonmyeloablative strategy for BM transplantation (BMT) as immunotherapy for minimal residual disease. MATERIALS AND METHODS: BALB/c mice inoculated with leukemia (BCL1) or breast carcinoma (4T1) cells were conditioned for BMT with TLI (200 cGy) followed by priming with donor (C57BL/6) BM cells on day 1, and by injection with 200 mg/kg cyclophosphamide on day 2. After conditioning (day 3), recipients were transplanted with BM cells from the same donor. Treated animals were monitored for 230 days for survival, development of leukemia/solid tumor, and GVHD. RESULTS: BMT converted the mice to complete chimeras and prevented development of leukemia in 90% of recipients and locally growing breast carcinoma in 40% of the mice. Immunization of donors of the second BM with 4T1 cells prevented development of breast carcinoma in 80% of 4T1 inoculated mice. Fewer animals treated for malignancy by nonmyeloablative BMT died of GVHD than those treated by myeloablative BMT. However, late GVHD-related mortality in mice treated for leukemia was higher than after nonmyeloablative BMT to naive recipients (p < 0.00001). Infusion of host-type anti-donor immune lymphocytes 8 days after BMT improved the survival of recipients treated for leukemia without affecting engraftment and the graft-vs-leukemia potential of donor BM. CONCLUSIONS: Effective eradication of malignant cells can be achieved following allogeneic BMT after nonmyeloablative conditioning.

Intraportal and systemic allogeneic cell therapy in a murine model of hepatic metastatic breast cancer.

Allogeneic immunocompetent splenocytes were tested for their ability to exert a GVT effect in a murine model of liver metastasis. Mammary carcinoma cells originating from an H-2(d) mouse were inoculated through the PV of F(1) (H-2(d/b)) mice, to mimic clinical hepatic involvement in malignant disease. Cell therapy was given either locally (PV) or systemically by IV inoculation to test differential efficacy of the GVT effect, and the differential expression of GVHD symptoms induced by diverse routes of administration. Livers of mice treated with H-2(b) derived splenocytes given PV or IV remained tumor-free for at least 4 weeks following tumor inoculation. Furthermore, all secondary recipients of adoptively transferred (AT) liver cells were tumor-free for >300 days. In contrast, all livers of untreated control mice or mice treated with syngeneic splenocytes displayed tumor metastases as early as 2 weeks following tumor inoculation, and large local tumors developed in AT secondary recipients. Our data demonstrate the efficacy of allogeneic cell therapy, given either locally or systemically, in the eradication of liver metastases. However, diverse routes of cell therapy administration did not show any difference in the expression and outcome of GVHD.

Cell Therapy With Preimmunized Effector Cells Mismatched for Minor Histocompatible Antigens in the Treatment of a Murine Mammary Carcinoma.

SUMMARY: Cell therapy with allogeneic donor cells mismatched for minor histocompatible (MiHC) antigens was applied to a murine mammary carcinoma (4T1) model to test the feasibility of graft versus tumor (GVT) effect against metastatic epithelial tumor cells. BALB/c mice bearing a 4T1 tumor of BALB/c origin were given syngeneic or MiHC-mismatched splenocytes. GVT effects were determined in secondary recipients of adoptively transferred lung cells derived from primary hosts who had previously been inoculated intravenously with 4T1 cells, and injected with one of the following: 1) naive BALB/c splenocytes, 2) naive DBA/2 splenocytes, 3) 4T1-immune DBA/2 splenocytes, or 4) DBA/2 splenocytes immunized with host-derived BABL/c spleen cells. Naive DBA/2 splenocytes inhibited tumor growth only slightly and only slightly prolonged the survival of secondary recipients, in comparison with fully matched tumor/host BALB/c spleen cells. An efficient GVT reaction was demonstrated in vitro and in vivo with MiHC-mismatched DBA/2 splenocytes from mice presensitized by multiple injections of irradiated tumor or BALB/c-derived spleen cells. All 30 mice adoptively inoculated with lung cells from primary hosts that had previously been treated with these presensitized effector cells were tumor free for >250 days. Secondary recipients inoculated with lung cells from mice given naive BALB/c or DBA/2 spleen cells died of metastatic tumors within 33 to 46 days. These results suggest that preimmunized donor cells represent an effective tool against metastatic disease; hence, the next goal should be to control graft-versus-host disease while exploiting the GVT potential.