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.

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.

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.

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.

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.

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.