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 gamma (IFNgamma). Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that IL-1beta and tumour necrosis factor alpha (TNFalpha) were produced by both cells. Higher pro-inflammatory cytokine (IL-1beta and TNFalpha) 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, TNFalpha or lipopolysaccharide (LPS) upregulated IL-12 p40 production by MAC-DCs, while IL-12 p70 remained undetectable. LPS stimulation also increased TNFalpha 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.

[Adoptive immunotherapy: evaluation and perspectives in the treatment of certain cancers]. / Immunothérapie adoptive: bilan et perspectives dans le traitement de certains cancers.

INTRODUCTION: Adoptive immunotherapy was first introduced in the 1980s. This new anticancer therapeutic approach has already demonstrated promising results in both animal models and humans affected by various tumors. CURRENT KNOWLEDGE AND KEY POINTS: This review summarizes the requirements of such therapies involving either activated lymphocytes, tumor-infiltrating lymphocytes or activated macrophages. It focuses more particularly on the promising approaches that represent antigen presenting cells such as macrophages and antigen-loaded dendritic cells in the development of safe and effective cancer vaccines. FUTURE PROSPECTS AND PROJECTS: Standardized procedures for macrophages and dendritic cell generation, as well as preliminary results of clinical applications in patients with either prostate cancer or melanoma, are also discussed.

Production of macrophage-activated killer cells for targeting of glioblastoma cells with bispecific antibody to FcgammaRI and the epidermal growth factor receptor.

OBJECTIVE: The aim was to determine the ability of macrophage-activated killer cells (MAK cells) obtained from peripheral blood of normal volunteers to kill glioblastoma multiforme (GBM) cell lines. Another goal was to investigate whether a bispecific antibody (bsAb) MDX-447, recognizing the high-affinity Fc receptor for IgG (FcgammaRI) and epidermal growth factor receptor (EGFR), would enhance MAK cell tumoricidal activity. METHODS: Monocytes, from leukapheresis product, were isolated by countercurrent elutriation and differentiated into MAK cells by culture with granulocyte/macrophage-colony-stimulating factor, vitamin D3 and interferon gamma. Cells were checked for sterility, endotoxin and phenotypic markers. MAK cell functional activity was measured by a flow-cytometric phagocytosis assay. Target cells, a carcinoma cell line and two glioma cell lines expressing EGFR, were stained with PKH-26. MAK cells were labeled with fluorescein-conjugated anti-CD14. Combined effectors, targets and bsAb were incubated and the percentage of MAK cells with phagocytosed targets was determined by flow cytometry. CONCLUSION: We demonstrate that a large number of highly purified monocytes, isolated from peripheral blood, can be differentiated into MAK cells for use as an adjuvant for cancer treatment. After culture these cells are sterile, endotoxin-free and comprise more than 95% MAK cells. Increased amounts of CD14, CD64 and HLA-DR, which are characteristics of macrophage activation, were expressed. MAK cells were extremely phagocytic in comparison to monocytes, even in the absence of bsAb. Moreover, bsAb enhanced the tumoricidal activity of elutriated MAK cells targeted against GBM cell lines. Therefore, intracavity MAK cells armed with MDX-447 could be an effective adoptive immunotherapy for EGFR-positive GBM.

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.

Monocyte-derived dendritic cells: development of a cellular processor for clinical applications.

Since dendritic cells (DCs) are the most professional antigen-presenting cells, (Schuler et al., 1997), increasing interest in their use in clinical approaches has been observed. (Nestle et al., 1998; Murphy G. et al., 1996). We have developed an ex vivo standardized process for the generation of dendritic-like cells (MAC-DCs) from human blood circulating monocytes. Human monocytes can differentiate into very different functional cells according to the conditions of culture, media and cytokines used. In the present study, we demonstrate that both pure monocytes and mononuclear cells differentiate into DCs when they are grown in defined medium AIM-V in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IL13 and in approved biocompatible non-adherent bags. Quality and functional controls of the immature DCs obtained rely on bacterial sterility, viability, morphology and recovery. The MAC-DCs also present an immature DC phenotype with a low expression of CD14 and CD64, and high expression of MHC-I, MHC-II and CD40. They also express B7 costimulatory molecules (CD80, CD86), CD83, and CD1a molecules. They induce strong allogenic T-cell proliferation (mixed lymphocyte reaction as well as proliferation of autologous memory T lymphocytes when incubated in the presence of recall antigens (tuberculosis, Candida albicans, and tetanus toxoid). They also show an increase in phagocytic uptake of yeast, tumour cells and debris. The global closed system which, under reproducible good medical practice (GMP) conditions, enables the production of dendritic cells of clinical quality, has been optimized ("Vac Cell Processor"). It contains all bags, connections, media, reagents, washing solutions, control antibodies, standard operating procedures, data management, traceability and help in the form of dedicated software.

Cellular vaccines.

This project is devoted to the development of novel cellular vaccines designed to treat cancer patients. These cellular vaccines present and enhance immunogens, which will elicit a potent immune response. The goal is to achieve safe and effective immune reaction against the patient's own tumour. (1) Autologous cellular vaccines are prepared by processing circulating blood mononuclear cells outside of the patient's body (ex vivo) to differentiate them into antigen-presenting cells (APCs). Monocyte-derived APCs (MD-APCs) are then grown in the presence of exogenous target antigens (tumour cell debris, or apoptotic bodies) to become fully mature APCs. (2) Functionality for antigen presentation to T cells of ex vivo MD-APCs is evaluated in vivo. (3) Cellular vaccines are tested in selected rodent animal models. Efficiency and immune response are monitored in pertinent experimental systems for cancer. Pharmacological data are generated for clinical investigation. Tolerance and biologic effects are documented in primates. (4) The first clinical trials on cancer patients are taking place in 1998 on melanoma and prostate cancer to validate the concept. Specialized cell processors with dedicated software and standardized controls are being developed and used for the preparation of cellular vaccines. (5) The evaluation of new non-viral vectors and the validation of new non-viral transfection methods of mononuclear cells with marker genes is in progress and will lead to the ex vivo transfection of genes coding for immunostimulating cytokines or for tumour antigens in MD-APCs. Efficiency will be validated in vitro and in animal models. The ex vivo and animal model studies validate the clinical relevance of this new cellular immunotechnology. Clinical validation of individual autologous cellular vaccines in specific indications for which no treatment is presently available will allow the development of cellular and gene immunotherapy for other types of cancers.

Immune therapy with macrophages: present status and critical requirements for implementation.

Adoptive transfer of activated macrophages has been validated in animal experimental tumor models; clinical trials are ongoing (70 patients up to now). The mechanisms involved are reviewed as well as improved and standardized procedures for macrophage differentiation and activation. New developments including specific Ag presentation and gene therapy are discussed.

Functional consequences of macrophage infection by human immunodeficiency virus: bispecific antibody targeting of HIV-1-infected cells to Fc gamma RI expressing effector cells.

Human monocytes/macrophages play a major role in pathogenesis of human immunodeficiency virus (HIV) infection. These cells have been suspected of acting as a reservoir for the virus and are important in viral dissemination and persistence in infected individual. Furthermore, several biologic and clinical features indicate that monocytes/macrophages from HIV-1-seropositive patients have characteristics of an activation status, including the ability to secrete high levels of cytokines. Dysregulation of the cytokine network may influence the level and the consequences of viral replication in infected monocytes/macrophages. Therefore, the development of virus-specific agents that may interfere with viral replication could help to slow down the fatal course of HIV infection. In this article, we try to further quantify the early and late kinetic patterns of the cytokine network during HIV-1 macrophage infection and report the biologic effects of virus-specific bispecific antibody (MDX-240) in HIV-1 macrophage infection.

In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody.

Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.

Antibody-dependent cellular cytotoxicity and neutralization of human immunodeficiency virus type 1 by high affinity cross-linking of gp41 to human macrophage Fc IgG receptor using bispecific antibody.

Human monocytes/macrophages, which express Fc receptors for IgG are involved in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis. These receptors are known to mediate numerous immunological functions including cell-mediated killing and possibly targeting of HIV to the lysophagosome monocyte-derived macrophage (MDM) entry route for virus neutralization. To study both activities in HIV-1 infection, MDM Fc gamma RI was specifically selected using bispecific antibody (Bs-Ab) containing whole human monoclonal antibody against gp41 and the Fab' fragment of murine anti-Fc gamma RI 22.2 antibody. Bs-Ab was found to mediate potent antibody-dependent cellular cytotoxicity and virus neutralization.

Bispecific anti-human red blood Rhesus-D antigen x anti Fc gamma RI targeted antibody-dependent cell-mediated cytotoxicity and phagocytosis by mononuclear leucocytes.

The Fc receptor mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by bispecific antibody (BsAb) to the high-affinity Fc receptor for IgG (Fc gamma RI) and to human red blood group antigen RhD were studied in vitro, using human mononuclear leucocytes as effector cells. The results were compared with those obtained by using a human monoclonal IgG1 anti-RhD used alone and a reference human polyclonal anti-RhD antibody. The effect of non-specific human IgG on FcR-mediated functions by mononuclear leucocytes was checked. The results demonstrate that BsAb presents a high resistance of Fc-mediated function to blockade by non-specific human IgG compared with that of both polyclonal and monoclonal anti-RhD antibodies. These results further encourage possible clinical application of bispecific antibody in passive immunotherapy.

Bispecific antibody targeting of human immunodeficiency virus type 1 (HIV-1) glycoprotein 41 to human macrophages through the Fc IgG receptor I mediates neutralizing effects in HIV-1 infection.

Human monocyte-derived macrophages that express the CD4 molecule and the Fc receptor for IgG (Fc gamma R) play a major role in the pathogenesis of human immunodeficiency virus (HIV) infection. To explore this possibility further, human monoclonal antibody to glycoprotein 41 (gp41) was produced, and a heterobifunctional antibody composed of F(ab') x F(ab')2 fragments of monoclonal anti-gp41 and anti-Fc gamma RI 22.2 were constructed. Both antibodies were analyzed for neutralizing effects, and the role of the CD4 molecule in HIV infection was studied with human monocyte-derived macrophages. The bispecific antibody exhibited strong neutralizing properties, in contrast to the monoclonal anti-gp41 antibody. Moreover, in the presence of monoclonal anti-Leu-3a antibody, viral production was completely inhibited. These findings demonstrate the necessity of the CD4 molecule in HIV infection of human macrophages and emphasize the usefulness of such heterobifunctional antibody directed to virus and monocyte-derived macrophage Fc receptors in prevention of HIV infection.

Functional consequences of monocyte/macrophage infection by HIV1.

Monocyte/macrophage infection by human immunodeficiency virus type 1 (HIV1) was studied for its effects on the production of tumour necrosis factor alpha (TNF alpha) and the expression of the manganese superoxide dismutase (MnSOD) gene. For this purpose, human peripheral blood monocytes were obtained from healthy HIV1-seronegative donors by centrifugal elutriation and infected with either the HIV1/LAV1 strain or with the primary HIV1/DAS isolate. The results showed that (1) HIV1/LAV1-infected macrophages did not produce any biologically detectable TNF alpha during the few hours following lentiviral infection, despite rises in the TNF alpha mRNA level; (2) MnSOD gene transcription in the macrophages increased, as measured 2 and 4 h after infection; (3) the level of the MnSOD gene expression declined during the late phases of lentiviral infection, but TNF alpha synthesis and gene expression rose; and (4) bispecific antibody comprised of anti-Fc gamma RI (anti-CD64) and anti-gp41 monoclonal antibodies inhibited the in vitro infection of monocyte-derived macrophages by HIV1/DAS.

Adoptive immunotherapy with bispecific antibodies: targeting through macrophages.

We report on two applications of bispecific antibodies to enhance the antitumoral function of human macrophages: (1) use of rhuIFN gamma (recombinant human IFN gamma) encapsulated in human red blood cells coated with anti-Fc gamma RI/anti-RhD+ bispecific antibodies to target and to activate human macrophages; encapsulated rhuIFN gamma was more potent than free IFN gamma in activating mature macrophages in vitro, demonstrating the efficacy of this delivery system to initiate in situ activation of macrophages and also to maintain a high antitumoral efficacy of macrophages with less side effects than after systemic injection of IFN gamma; (2) targeting of activated macrophages to tumours by bispecific antibodies directed against macrophage Fc gamma RI and against human adenocarcinoma antigen; differentiated human macrophages became cytotoxic for human adenocarcinoma in vitro and in vivo (tumours implanted in nude mice) when activated by rhuIFN gamma; this effect was increased in the presence of bispecific antibodies. These two approaches were aimed at increasing the efficacy of cellular immunotherapies using activated macrophages as effector cells (macrophage-activated killer, or MAK), an adoptive therapy which we have developed. Bispecific antibodies could increase specific homing and activation of cytotoxic MAK effectors at tumour sites.

Fc receptors for IgG (Fc gamma Rs) on human monocytes and macrophages are not infectivity receptors for human immunodeficiency virus type 1 (HIV-1): studies using bispecific antibodies to target HIV-1 to various myeloid cell surface molecules, including the Fc gamma R.

Fc gamma Rs (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) are highly expressed on human mononuclear phagocytes and function in the clearance of immune complexes and opsonized pathogens. We have examined the role of Fc gamma R in mediating antibody-dependent clearance of HIV-1 by human monocytes and monocyte-derived macrophages by using bispecific antibodies (BsAbs) to independently target the virus to Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Virus production was markedly reduced in monocytes cultured with strain HIV-1IIIB opsonized with BsAbs that target the virus to either Fc gamma RI or Fc gamma RII compared to monocytes cultured with virus in the absence of BsAbs or in the presence of BsAbs that target the virus to non-Fc gamma R surface antigens (CD33 and HLA-A,B,C). These results were confirmed using the monotropic isolate HIV-1JRFL. Interaction of HIV-1JRFL with Fc gamma RI or Fc gamma RII on human monocytes and Fc gamma RI, Fc gamma RII, or Fc gamma RIII on monocyte-derived macrophages resulted in markedly reduced levels of virus production in these cultures. Moreover, HIV-1 infection of monocytes and monocyte-derived macrophages was completely blocked by anti-CD4 monoclonal antibodies, indicating that interaction with CD4 is required for infectivity even under conditions of antibody-mediated binding of HIV-1 to Fc gamma R. Thus, we propose that highly opsonized HIV-1 initiates high-affinity multivalent interactions with Fc gamma R that trigger endocytosis and intracellular degradation of the antibody-virus complex. At lower levels of antibody opsonization, there are two few interactions with Fc gamma R to initiate endocytosis and intracellular degradation of the antibody-virus complex, but there are enough interactions to stabilize the virus at the cell surface, allowing antibody-dependent enhancement of HIV-1 infection through high-affinity CD4 interactions. However, our results suggest that interaction of highly opsonized HIV-1 with Fc gamma Rs through BsAbs may reduce viral infectivity through Fc gamma R-mediated cytotoxic mechanisms and, therefore, that BsAbs offer promise as therapeutic reagents in HIV-1 infections.

Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain.

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.

Bispecific antibodies and targeted cellular cytotoxicity.

The Second International Conference on Bispecific Antibodies (BsAbs) and Targeted Cellular Cytotoxicity considered how targeted cytotoxicity can be used (1) to increase understanding of the general mechanisms of cellular cytotoxicity and (2) clinically in the treatment of cancer and infectious disease. BsAbs, the main mediators of targeted cellular cytotoxicity, can be made by chemical crosslinking, by fusing hybridoma cells and by molecular genetic approaches. BsAbs bind to target cells via one V region and trigger molecules such as T-cell receptors (TCRs) or FcR for IgG (Fc gamma R) on cytotoxic cells via their other V region. This linking of triggering structures to target cells induces target cell lysis and provides important clues to the signals used to elicit the lytic process.

Hepatitis B virus core antigen (HBc Ag) accumulation in an HBV nonproducer clone of HepG2-transfected cells is associated with cytopathic effect.

Two clones of the hepatoblastoma HepG2 cell line transfected with complete hepatitis B virus deoxyribonucleic acid (HBV DNA) were studied. The kinetics and cytopathic effect of HBV Ag production in these two clones (one of which was an HBV producer) were compared to those of the parent HepG2 cell line. The presence of hepatitis B surface antigen (HBs Ag) and hepatitis B e antigen (HBe Ag) was determined by commercial enzyme-linked immunosorbent assay (ELISA). A hepatitis B core antigen (HBc Ag)-specific ELISA assay was developed, using monoclonal anti-HBc to detect HBc Ag. Amounts of HBs, HBe, and HBC Ags were partially quantified in both intracellular and extracellular compartments. The HBV producer clone excreted high levels of HBc, HBe, and HBs Ags from the beginning of the growth phase, and no cytopathic effect was observed. The HBV nonproducer clone produced high levels of HBs and HBe Ags, but there was no detectable HBc Ag in the supernatant; instead, HBc Ag accumulated in the intracellular compartment. In this clone, significant cell death was observed 4 days after cell confluency, corresponding with notable HBc Ag release into the supernatant. These results suggest a cytopathic effect associated with HBc Ag accumulation in the HBV nonproducer clone, but no cytopathic effect in the HBV producer clone. This suggests that virological factors as well as the host's immune response may be considered in explaining liver injury occurring in hepatitis B.

Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers.

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10(7) - 2 x 10(7) cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.