BACKGROUND AND OBJECTIVES Cytokine-induced killer (CIK) cells have shown anti-leukemic activity and little graft-versus-host disease (GVHD) in several animal models. The safety of these cells in autologous settings has been shown. We performed a phase I study of allogeneic (donor's) CIK cells in patients relapsing after allogeneic haematopoietic stem cell transplantation (HSCT). DESIGN AND METHODS Eleven patients with acute myelogenous leukemia (n=4), Hodgkin's disease (n=3), chronic myelomonocytic leukemia, (n=1), pre-B acute lymphoblastic leukemia (n=1) and myelodysplasia (n=2), all of whom had relapsed after sibling (n=6) or matched unrelated donor (n=5) HSCT, entered this study. RESULTS Before CIK administration, six patients had received other salvage treatments including chemotherapy (n=5), radiotherapy (n=1) and unmanipulated donor lymphocytes (n=6) without any significant tumor response. The median number of CIK infusions was two (range 1-7) and the median number of total CIK cells was 12.4x106/kg (range 7.2-87.4). The infusions were well tolerated and no acute or late infusion-related reactions were recorded. Acute GVHD (grade I and II) was observed in four patients, 30 days after the last CIK infusion, and progressed into extensive chronic GVHD in two cases. Disease progression and death occurred in six patients. One patient had stable disease, one had hematologic improvement and three achieved complete responses. INTERPRETATION AND CONCLUSIONS This study shows that the production of allogeneic CIK cells is feasible under clinical-grade conditions, well tolerated and may contribute to clinical responses.

Objective. Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells.Materials and Methods. Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (M), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2).Results. Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL, patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern.Conclusions. Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

We have used a standardized 21-day expansion protocol to produce cytokine-induced killer (CIK) cells starting from very small amounts of nucleated cells (approximately 15 × 10 6 cells) isolated from cord blood. Mononuclear cells are stimulated with anti CD3 (OKT3) and IFN γ and then expanded with IL-2. Moreover, we show that washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500 × 10 6 CIK by the same expansion protocol. CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels. The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions and therefore these observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.

Cytokine induced killer cells (CIK) are T/NK cells with demonstrated anti-tumoral activity but lack of GVHD reactivity. They are expanded in vitro after stimulation of PBMC with OKT3, IFN-γ and rhIL-2. This phase I study was designed to test the safety and feasibility of repeated infusions of in vitro expanded donor derived CIK cells given to patients relapsed after allogeneic HSCT. The mean number of starting total nucleated cells was 707 × 106 (range 58–1500 × 106). After a median 22 days of culture, a mean percentage of 51% CD3+CD56+ cells (range 40–71%) was obtained corresponding to an absolute mean number of 1421×106 total CIK cells (range 422–2470 × 106). Eleven patients with AML (n=4), HD (n=3), CMML (n=1), pre-B ALL (n=1) and MDS (n=2), all relapsed after sibling (6) or matched unrelated donor (5) HSCT, entered this study. Before CIK administration, 7 patients had received one or more additional salvage treatments including chemotherapy (5), radiotherapy (1) and unmanipulated DLI (6) without any significant tumor response. The median number of CIK infusions was 2 (range 1–7) and the median number of total CIK cells was 14.5 ×106/kg (7.2–51). The infusions were well tolerated and no acute or late infusion-related reactions were registered. Acute GVHD (grade I and II) was observed in 4 patients 30 days after the last CIK infusion, which progressed into extensive chronic GVHD in 2 cases. In 6 patients, no significant clinical response could be registered so that disease progression and death occurred rapidly. In contrast, 5 patients achieved measurable responses: a patient with MDS, who had been treated with CIK cells alone, showed a hematologic improvement but subsequently progressed and died. One patient with HD received local radiotherapy and 7 CIK infusions (total of 51×106/kg CIK cells) which allowed the achievement of a good PR. After almost 1 year (300 days), he progressed and chemotherapy was given with achievement of a very good PR. A second patient with HD received one DLI at day 516 and 1 CIK infusion (12.2×106/kg) at day 537. At day 572, chemotherapy was initiated due to the persistence of disease. At the end of chemotherapy he received 3 additional CIK infusions for a total of 34×106/kg from days 711–752, without signs of aGVHD and is presently in CR at more than 780 days. One patient with CMML had been treated with DLI on day 102 and with a total of 38×106/kg CIK cells, given in four infusions from days 137–530, because of the appearance of mixed chimerism. Full chimerism was achieved 42 days after the first CIK infusion. The patient remains in CR at day 576. A second MDS patient, who had not achieved any significant response after five DLI (days 411–559), obtained a complete hematologic, cytogenetic and molecular remission after a single CIK infusion (7.6 ×106/kg) given at day 603 and remains in CR at day 680. This study shows that the production of allogeneic CIK cells is feasible, their infusion is generally safe and may induce clinical remission in patients relapsing after HSCT.