Jean-Loup Romet-Lemonne

Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties.

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

Adoptive Immunotherapy For Superficial Bladder Cancer With Autologous Macrophage Activated Killer Cells

PURPOSE We assessed the efficacy and safety of adoptive immunotherapy administered to 17 patients with TaGIII or recurrent TaGII superficial bladder cancer following transurethral tumor resection. MATERIALS AND METHODS Macrophage activated killer (MAK) cells were obtained from autologous mononuclear cells harvested by apheresis, after in vitro culture for 7 days and activation with interferon-gamma on the last day of culture. The patients received 6 weekly intravesical infusions of approximately 2 x 10(8) cells each. Additionally, 5 patients received 2 or 3 more infusions at 3-month intervals. Each patient was followed for 1 year or until tumor recurrence, whichever came first. RESULTS A total of 112 intravesical infusions were performed. During the 12-month followup period 8 patients experienced 11 common toxicity criteria grade 1 or grade 2 adverse events considered possibly related to protocol. No clinically relevant grade 1 or 2 laboratory test results were reported while the patients received treatment. In 17 patients 8 tumors recurred compared to 34 recurrences during the year before the first MAK cell infusion. This difference was highly significant (p </=0.0005). CONCLUSIONS The promising efficacy and safety results of this study and the fact that the MAK cell treatment regimen proved feasible should encourage initiation of further large scale studies to confirm these data.

Cytokine production and T-cell activation by macrophage-dendritic cells generated for therapeutic use.

Clinical grade ex vivo‐generated antigen‐presenting cells, macrophage–dendritic cells (MAC–DCs) or macrophage‐activated killers (MAKs) were derived from peripheral blood mononuclear cells (PBMCs). Cultures (7 d) were performed in non‐adherent conditions in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and either interleukin 13 (IL‐13) or dihydroxy‐vitamin D3 respectively. MAKs were activated during the last 24 h with interferon γ (IFNγ). Reverse transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) analyses indicated that IL‐1β and tumour necrosis factor α (TNFα) were produced by both cells. Higher pro‐inflammatory cytokine (IL‐1β and TNFα) amounts were detected on average in MAK supernatants. In contrast, IL‐12 p40 was found only in MAC–DC supernatants, but the biologically active IL‐12 form (p70) was never detected. T‐cell cytokines (IL‐2, IL‐4, IL‐10) were not produced in culture conditions in which T cells were nevertheless present. At d 7, TNFα or lipopolysaccharide (LPS) upregulated IL‐12 p40 production by MAC–DCs, while IL‐12 p70 remained undetectable. LPS stimulation also increased TNFα production by these cells. Allogeneic mixed lymphocyte reactions (MLR) showed that MAKs are poor stimulatory cells compared with MAC–DCs. The MAC–DC stimulatory capacity was enhanced by LPS, although the expression of HLA class II, CD83, CD80 and CD86 was unmodified. Thus, MAC–DCs represent a tool for triggering adaptative immunity, while MAK should be primarily used as effector killer cells.

Autologous activated macrophages (MAK™) coated ex vivo with humanized anti‐CD20 monoclonal antibodies can eradicate minimal residual disease in chronic lymphocytic leukaemia in clinical response

Heavily pretreated chronic lymphocytic leukaemia (CLL) is associated with poor survival, as responses to the last treatment received are short-lived. The response duration correlates with the quality of remission, i.e. the level of persistent minimal residual disease (MRD) (Brugiatelli et al, 1997). We sought to improve the response duration by reducing MRD in such heavily treated patients achieving a new response. We therefore conducted a phase II trial of autologous activated macrophages (MAKs) coated with a humanized anti-CD20 antibody (rituximab), focusing on its safety and its capacity to eradicate MRD. Ten patients with Matutes score 5 CLL were enrolled. Five of the seven patients tested had wild-type IGHV (Table I). The patients had received between 2 and 6 previous lines (median 4) of chemotherapy, including at least one fludarabine-based regimen. Moreover, seven patients had been autografted after conditioning with total body irradiation and cyclophosphamide. Before inclusion in the study all the patients were treated until they reached their best possible response, based on the National Cancer Institute Working Group (NCI-WG) criteria. At inclusion, six patients were in complete remission (CR) and four had a partial response (PR). All the patients had MRD (presence of residual leukaemic cells), as demonstrated with a 4-colour phenotypic assay and polymerase chain reaction (PCR) amplification of the clonal immunoglobulin gene. Patients’ peripheral blood mononuclear cells (PBMC; 10) were isolated with a COBE Spectra device. Macrophages were produced from the PBMC with the MAK cell processor (Immuno-Design-Molecules, Paris, France) (Goxe et al, 1998). After 6 d of culture, interferon-c (Boehringer Ingelheim, France) was added at a concentration of 250 IU/ml to activate macrophages. On day 7, MAKs were purified by elutriation (B. Braun, France) and resuspended in 200 ml of serum albumin in the presence of 2Æ5 mg of rituximab (Roche, Basel, Switzerland). The number of MAKs to be reinfused was based on previously published experience (Faradji et al, 1991). The dose of rituximab (2Æ5 mg) was calculated in order to saturate immunoglobulin constant-region-fragment cell-surface receptors (FcR) of MAKs. Six infusions of MAKs/anti-CD20 were planned at one-week intervals. The primary endpoint was a decrease in MRD, as shown by a negative four-colour flow cytometric immunophenotype (Maloum et al, 2002) and confirmed by clone-specific PCR (cPCR).