We treated 93 patients who had acute nonlymphoblastic leukemia in the first remission or chronic myelocytic leukemia in the chronic phase (median age, 30 years) with high-dose cyclophosphamide and fractionated total-body irradiation, followed by infusion of marrow from an HLA-identical sibling. To evaluate postgrafting prophylaxis for graft versus host disease, we studied these patients in a sequential, prospective, randomized trial that compared the effect of a combination of methotrexate and cyclosporine (n = 43) with that of cyclosporine alone (n = 50). All patients had evidence of sustained engraftment. A significant reduction in the cumulative incidence of grades II to IV acute graft versus host disease was observed in the patients who received both methotrexate and cyclosporine (33 percent), as compared with those who were given cyclosporine alone (54 percent) (P = 0.014). Seven patients who received cyclosporine alone acquired grade IV acute graft versus host disease, as compared with none who received both methotrexate and cyclosporine. Thirty-five of the 43 patients given both methotrexate and cyclosporine and 31 of the 50 patients given cyclosporine are alive as of this writing, at 4 months to 2 years (median, 15 months); the actuarial survival rates in the two groups at 1.5 years were 80 percent and 55 percent, respectively (P = 0.042). We conclude that the combination of methotrexate and cyclosporine is superior to cyclosporine alone in the prevention of acute graft versus host disease after marrow transplantation for leukemia, and that this therapy may have a beneficial effect on long-term survival.

Three patients had leukocytosis of large granular lymphocytes and chronic neutropenia. Clonal chromosomal abnormalities (trisomy 8 and trisomy 14) and lymphocytic infiltration of splenic red pulp, hepatic sinusoids, and bone marrow indicated the neoplastic nature of the large granular lymphocytes. Demonstration of a T3+, T8+, HNK-1 + phenotype and low natural killer cell activity that was augmented by interferon treatment showed the leukemic cells to be immature natural killer cells. Multiple autoantibodies were present and included rheumatoid factor and antinuclear, antineutrophil, antiplatelet, and antierythrocyte antibodies, suggesting a defect of B-cell immunoregulation. In addition, in-vitro studies showed impaired suppression of immunoglobulin biosynthesis by abnormal cells from one patient. Antineutrophil antibodies and absence of direct cell-mediated inhibition of granulocyte-macrophage colony formation supported a humoral immune mechanism for the neutropenia. In these patients the syndrome of splenomegaly, multiple autoantibodies with neutropenia, and lymphocytosis of large granular lymphocytes is due to a neoplastic proliferation of immature natural killer cells.

Background-Treatment of relapsed hematological malignancies after an allogeneic peripheral blood stem cell transplant (SCT) is challenging. Donor lymphocyte infusion (DLI) from the stem cell donor is an attractive clinical option to salvage this group of patients.

PURPOSE This study reports a phase I immunotherapy (IT) trial in 23 women with metastatic breast cancer consisting of eight infusions of anti-CD3 × anti-HER2 bispecific antibody (HER2Bi) armed anti-CD3 activated T cells (ATC) in combination with low dose interleukin 2 (IL-2) and granulocyte-macrophage-colony stimulating factor to determine safety, maximum tolerated dose (MTD), technical feasibility, T cell trafficking, immune responses, time to progression, and overall survival (OS). EXPERIMENTAL DESIGN ATC were expanded from leukapheresis product using IL-2 and anti-CD3 monoclonal antibody and armed with HER2Bi. In 3+3 dose escalation design, groups of 3 patients received 5, 10, 20, or 40 × 109 armed ATC (aATC) per infusion. RESULTS There were no dose limiting toxicities and the MTD was not defined. It was technically feasible to grow 160 × 109 ATC from a single leukapheresis. aATC persisted in the blood for weeks and trafficked to tumors. Infusions of aATC induced anti-breast cancer responses and increases in immunokines. At 14.5 weeks after enrollment, 13 of 22 (59.1%) evaluable patients had stable disease and 9 of 22 (40.9%) had progressive disease. The median OS was 36.2 months for all patients, 57.4 months for HER2 3+ patients, and 27.4 months for HER2 0–2+ patients. CONCLUSIONS Targeting HER2 positive and negative tumors with aATC infusions induced anti-tumor responses, increases in Th1 cytokines and IL-12 serum levels that suggest that aATC infusions vaccinated patients against their own tumors. These results provide a strong rationale for conducting phase II trials.

Relapse after adjuvant chemotherapy or high-dose chemotherapy with stem cell transplant for high-risk breast cancer remains high and new strategies that provide additional antitumor effects are needed. This report describes methods to generate highly effective HER2/neu-specific cytotoxic T cells by arming activated T cells with anti-CD3 x anti-HER2/neu bispecific antibody (BsAb). OKT3 and 9184 (anti-HER2) monoclonal antibodies (mAb) were conjugated and used to arm T cells that were subsequently tested in binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregated and specifically killed HER2/neu(+) breast cancer cells. Cytotoxicity emerged after 6 days of culture, was higher in armed T cells than unarmed T cells at all effector to target ratios (E/T) tested, and increased as the arming dose was increased. At an E/T of 20:1, the mean cytotoxicity of armed activated T cells (ATC) from 10 normal subjects increased by 59 +/- 11% (+/-SD) over that seen in unarmed ATC (p < 0.001) and the mean cytotoxicity of armed ATC from 6 cancer patients increased by 32 +/- 9% above that seen for unarmed ATC (p < 0.0004). After arming, the BsAb persisted on ATC up to 72 h and armed ATC continued to be cytotoxic up to 54 h. The amount of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted was 1699, 922, and 3092 pg/ml/10(6) cells per 24 h, respectively, when armed T cells were exposed to a HER2/neu(+) breast carcinoma cell line. These studies show the feasibility and clinical adaptability of this approach for generating large numbers of anti-HER2-specific, cytotoxic T cells for clinical trials.

Asthma is a worldwide health problem. Activated T cells (ATCs) in the lung, particularly T helper 2 cells (Th2), are strongly associated with inducing airway inflammatory responses and chemoattraction of inflammatory cells in asthma. Small interfering RNA (siRNA) as a promising anti-sense molecule can specifically silence inflammation related genes in ATCs, however, lack of safe and efficient siRNA delivery systems limits the application of siRNA as a therapeutic molecule in asthma. Here, we designed a novel pulmonary delivery system of siRNA, transferrin-polyethylenimine (Tf-PEI), to selectively deliver siRNA to ATCs in the lung. Tf-PEI polyplexes demonstrated optimal physicochemical properties such as size, distribution, zeta-potential, and siRNA condensation efficiency. Moreover, in vitro studies showed significantly enhanced cellular uptake and gene knockdown mediated by Tf-PEI polyplexes in human primary ATCs. Biodistribution of polyplexes in a murine asthmatic model confirmed that Tf-PEI polyplexes can efficiently and selectively deliver siRNA to ATCs. In conclusion, the present work proves the feasibility to target ATCs in asthma via Tf receptor. This strategy could potentially be used to design an efficient siRNA delivery system for asthma therapy.

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Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 F 0.3% to 89.4 F 2.1% at an E:Tratio of100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8 + NKG2D + CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.