The anti-CD20 mAb LFB-R603 interrupts the dysregulated NF-κB/Snail/RKIP/PTEN resistance loop in B-NHL cells: role in sensitization to TRAIL apoptosis.

LFB-R603 is a chimeric anti-CD20 mouse/human monoclonal antibody with a human IgG1 constant (Fc) region. In comparison to rituximab, LFB-R603 exhibits a high affinity to the Fcγ RIII (CD16) and a potent in vitro antibody-dependent cellular cytotoxicity (ADCC). We examined several experimental designs for the biological anti-tumor activity of LFB-R603 as well as its sensitizing activity to apoptosis in resistant non-Hodgin's B-cell lymphoma (B-NHL) in comparison to rituximab. Treatment of the B-NHL cell line Ramos with LFB-R603 was not toxic at the concentrations used and induced cell aggregation following culture at 24 and 48 h similarly to rituximab. Hence, we hypothesized that LFB-R603 may signal the tumor cells and modify intracellular survival/anti-apoptotic pathways. Treatment of Ramos cells with LFB-R603 inhibited the constitutively active NF-κB survival pathway, followed by significant potentiation of TRAIL-mediated apoptosis. We examined the underlying molecular mechanism by which the LFB-R603-mediated NF-κB inhibition results in the reversal of tumor cell resistance to TRAIL. We hypothesized that downstream gene products regulated by NF-κB that are involved in the resistance may be involved in LFB-R603-mediated sensitization. We found that LFB-R603 inhibited NF-κB activation and the anti-apoptotic factor Snail and induced the pro-apoptotic factor RKIP. The direct roles of Snail and RKIP modulation by LFB-R603 in the reversal of B-NHL resistance to TRAIL were examined by knocking down Snail and overexpressing RKIP in Ramos cells, respectively. Both approaches increased significantly the cell sensitivity to TRAIL apoptosis. In addition to changes observed in the expression levels of Snail and RKIP, Ramos cells treated with LFB-R603 induced the expression of PTEN along with inhibition of the PI3K-AKT activated pathway. PTEN silencing in Ramos cells pretreated with LFB-R603 and TRAIL inhibited TRAIL apoptosis; thus, demonstrating that PTEN induction by LFB-R603 has a direct role in tumor cell sensitization to TRAIL apoptosis. Several of the findings obtained in the experimental designs with LFB-R603 were superior to those obtained with rituximab. Overall, the findings demonstrate that LFB-R603 interferes with the dysregulated NF-κB/Snail/RKIP/PTEN/AKT resistance circuitry in B-NHL cells. Further, the findings suggest that LFB-R603 may sensitize tumor cells to various apoptotic stimuli including cytotoxic ligands such as TRAIL and chemotherapeutic drugs.

Analysis and characterization of antitumor T-cell response after administration of dendritic cells loaded with allogeneic tumor lysate to metastatic melanoma patients.

The primary goal of cancer vaccines is to induce CD8+ T cells specific for tumor-associated antigens (TAA) but the characterization of these cells has been difficult because of the low sensitivity of ex vivo assays. Here, we focused on TAA-specific CD8+ T-cell responses in melanoma patients after vaccination with autologous dendritic cells loaded with lysates derived from allogeneic tumor-cell lines (Lysate-DC). Out of 40 patients treated, 16 patients developed immune response to tumor-cell lysate and/or CD8+ T cells specific for differentiation and cancer-testis antigens. TAA-specific CD8+ T-cell responses were detected by interferon (IFN)-gamma enzyme-linked immunospot after in vitro sensitization and were, either transient during the treatment period or delayed, that is, observed after completion of all vaccinations. We could not correlate these immune responses to clinical data as none of the patients achieved an overall objective response according to Response Evaluation Criteria in Solid Tumors criteria. Three patients were reported as stable disease and 10 patients presented evidence of antitumor activity. We found that TAA-specific T cells characterized in 4 patients produced perforin ex vivo, but no IFN-gamma in enzyme-linked immunospot. Differential expression of IFN-gamma and perforin was also observed for viral-specific T cells. Altogether, our results show that Lysate-DC therapy elicited tumor-specific CD8+ T cells nonlimited to human leukocyte antigen-A2+ patients, with some T cells secreting perforin ex vivo and IFN-gamma only after restimulation. The differential expression of perforin and IFN-gamma by antitumor and antiviral CD8+ T cells supports that the sole use of IFN-gamma production to monitor T cells overlooks functional T-cell subpopulations triggered by vaccines.

Induction of multiple CD8+ T cell responses against the inducible Hsp70 employing an Hsp70 oligoepitope peptide.

The inducible heat shock protein Hsp70 has been described as a tumour antigen being frequently overexpressed in tumours of various histologic origins, with a role in tumourigenicity, as a critical event in tumour progression. A strategy to enhance the immune response to an antigen is the identification of multiple epitopes and the induction of a polyspecific response. Applied to tumour vaccination, such a polyspecific response should lead to a more robust antitumour efficacy. The long peptide Hsp70380-402 encompasses three nonamer peptides with a high affinity for HLA-A *0201. In a previous paper, we have shown that two of these nonamer peptides, p391 and p393, can raise CTL to recognize tumour cells overexpressing Hsp70. In the present paper, we demonstrate that the third nonamer peptide, p380, is a new epitope efficient in raising an antitumour immune response. The p380-402 polypeptide was able to induce an immune response against each of the three constituent epitopes both in vivo in HLA-A *0201 transgenic mice and in vitro with human PBMC. This polypeptide therefore constitutes an interesting candidate for the induction of multiple HLA-A *0201-restricted anti-Hsp70 antitumour CTL responses.

Involvement of TBK1 and IKKepsilon in lipopolysaccharide-induced activation of the interferon response in primary human macrophages.

Interferon (IFN) is an important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor-dependent and -independent mechanisms. In human macrophages and macrophage-activated killer cells, we demonstrate that (i) the type I IFN response to lipopolysaccharide (LPS) is weak compared to the host response to virus infection; (ii) there is a temporal difference in the induction of tank-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-related kinase epsilon (IKKepsilon) kinase activities in response to LPS, with TBK1 activated early and IKKepsilon induced in the late phase of IFN induction; and (iii) interferon regulatory factor (IRF)-7 is induced following LPS treatment, but there is no evidence that IRF-7 becomes activated by phosphorylation in vivo. Specifically, TBK1 kinase activity is rapidly increased after LPS stimulation (15 min) whereas IKKepsilon activation occurs at 8 h. RNA interference-mediated inhibition of TBK1 and IKKepsilon expression in macrophages interfere with IFNB and IRF7 gene expression following LPS activation. Macrophage priming with rIFN-alpha increased IRF-7 expression, led to a sharp up-regulation of the IFNB gene and to a rapid induction of IFNA2 upon LPS stimulation. These data support a differential role of TBK1 and IKKepsilon in the downstream response mediated by IRF-3 and IRF-7 to LPS in primary human macrophages.

Distinct roles for IFN regulatory factor (IRF)-3 and IRF-7 in the activation of antitumor properties of human macrophages.

When properly activated, macrophages can be tumoricidal, thus making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-gamma-activated macrophages, produced in large scale for clinical use (MAK cells), have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IFN regulatory factor (IRF)-3 and IRF-7, with subsequent type I IFN production, may be involved in the acquisition of new antitumor functions by macrophages. Adenoviral vectors were generated for the delivery of constitutively active forms of IRF-3 (Ad-IRF-3) or IRF-7 (Ad-IRF-7) into primary human macrophages. Cell death was observed in Ad-IRF-3-transduced macrophages, whereas Ad-IRF-7-transduced macrophages produced type I IFNs and displayed increased expression of genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand, interleukin (IL)-12, IL-15, and CD80, persisting for at least 96 hours. Expression of iNOS, TNF-alpha, FasL, IL-1, and IL-6 genes was unaltered by Ad-IRF-7 transduction. Interestingly, Ad-IRF-3 or Ad-IRF-7 transduction negatively regulated the transcription of protumorigenic genes encoding vascular endothelial growth factor and matrix metalloproteinase-2. Furthermore, Ad-IRF-7-transduced macrophages exerted a cytostatic activity on different cancer cell lines, including SK-BR-3, MCF-7, and COLO-205; the latter cells were shown previously to be insensitive to MAK cells. In conclusion, transduction of active forms of IRF-3 or IRF-7 differentially modulate the apoptotic and antitumor properties of primary macrophages, with active IRF-7 leading to the acquisition of novel antitumor effector functions.

Ex vivo-activated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibody-dependent cellular cytotoxicity and impact of human serum.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the mechanisms of tumor killing during antibody (Ab) immunotherapy, and a role for myeloid cells as effectors has been observed in several models. We are developing immunotherapy approaches based on administration of large numbers of ex vivo interferon-gamma-activated macrophages to cancer patients. With a quantitative assay measuring killing of nonproliferating tumor cells, we evaluated whether, in physiologic conditions, these macrophages synergize with the anti-CD20 Ab rituximab for killing primary B-cell chronic lymphocytic leukemia (B-CLL) cells. ADCC reached levels of 70% to 80% at effector to target ratios as low as 1:1. Macrophage recruitment by Ab-opsonized tumor cells did not result in enhanced cytokine secretion, suggesting that the cytokine shower observed in rituximab-treated patients is not caused by macrophage activation, and that cytokines have no role in CLL killing. We observed that uptake of tumor material by macrophages was not directly correlated to tumor killing. Nonetheless, experiments in the presence of cytochalasin D showed that ADCC occurred mainly by phagocytosis. Tumor killing was largely mediated by Fc gammaRI and inhibited by increasing concentration of serum. Importantly, complement deposition on B-CLL cells did not seem to enhance macrophage ADCC in this model, as complement-depleted and complement-repleted human plasmas exerted comparable inhibition.

Dendritic cells and HIV-specific CD4+ T cells: HIV antigen presentation, T-cell activation, and viral transfer.

Human immunodeficiency virus (HIV)-specific CD4+ lymphocytes are preferentially infected in HIV-positive individuals. To study this preferential infection, we have derived several HIV-specific (HS) CD4+ clones. We show that in dendritic cells (DCs), HIV virion capture led to major histocompatibility complex class-II (MHC-II)-restricted viral antigen presentation and to activation of HS cells. In contrast, neither cell-free virions nor infected lymphocytes activated HS cells. In DCs, the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN/CD209), which internalizes virions, promoted MHC-II presentation of HIV antigens. Activation of HS cells by HIV-exposed DCs triggered an efficient viral spread in lymphocytes. CD4+ clones with irrelevant antigenic specificities were not activated by HIV-exposed DCs and poorly supported viral replication under this setting. Our results unravel the mechanisms of MHC-II-restricted HIV antigen presentation by DCs and describe how HIV gains access to the very cells designed by the immune system to counteract this pathogen.

Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate.

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation.

Anti-tumor properties of human-activated macrophages produced in large scale for clinical application.

When properly activated, macrophages can be tumoricidal. To harness the therapeutic potential of these cells, we have developed a process for ex vivo production of large numbers of IFN-gamma-activated monocyte-derived macrophages. These monocyte-derived activated killer (MAK) cells have been safely administered to cancer patients with minimal residual disease in phase I/II clinical studies. To evaluate efficacy of treatment with MAK cells, phase III clinical studies are necessary. The process of MAK cell production has been further optimized and qualified for use in large cohorts of patients. In this study, we characterized MAK cells produced in large scale by studying their phenotype and functions. MAK cells were shown to exert anti-tumor activity by killing tumor cells and inhibiting their proliferation. These activities were enhanced by activation with IFN-gamma and addition of anti-tumor antibodies. Tumor necrosis factor-alpha (TNF-alpha) was one of the mediators used by MAK cells to inhibit tumor proliferation. To facilitate logistics of clinical trials, a process for MAK cell cryopreservation has been developed. We verified in vitro that cryopreserved cells retained the activity of fresh cells and were stable during storage. The safety and efficacy of cryopreserved MAK cells (Bexidem) are currently being assessed on superficial bladder cancer patients in a phase II/III clinical trial.

Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector.

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.

IL-2 production by virus- and tumor-specific human CD8 T cells is determined by their fine specificity.

Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-gamma, whereas effector memory CD8 cells (CD45RAdullCCR7-) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-gamma. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.

Vaccination of prostatectomized prostate cancer patients in biochemical relapse, with autologous dendritic cells pulsed with recombinant human PSA.

This study was conducted in prostate cancer patients in biochemical relapse after radical prostatectomy, to assess the feasibility, safety, and immunogenicity of therapeutic vaccination with autologous dendritic cells (DCs) pulsed with human recombinant prostate-specific antigen (PSA) (Dendritophage-rPSA). Twenty-four patients with histologically proven prostate carcinoma and an isolated postoperative rise of serum PSA (>1 ng/ml to 10 ng/ml) after radical prostatectomy were included. The patients received nine administrations of PSA-loaded DCs by combined intravenous, subcutaneous, and intradermal routes over 21 weeks. Postbaseline blood tests were performed at months 1, 3, 6, 9, and 12 (PSA levels), at months 6 and 12 (circulating prostate cancer cells), at month 6 (anti-PSA IgG and IgM antibodies), and at up to eight time points before, during, and after immunization (PSA-specific T cells). Circulating prostate cancer cells detected in six patients at baseline were undetectable at 6 months and remained undetectable at 12 months. Eleven patients had a postbaseline transient PSA decrease on one to three occasions, predominantly occurring at month 1 (7 patients) or month 3 (2 patients). Maximum PSA decrease ranged from 6% to 39%. PSA decrease on at least one occasion was more frequent in patients with low Gleason score ( p=0.016) at prostatectomy and with positive skin tests at study baseline ( p=0.04). PSA-specific T cells were detected ex vivo by ELISpot for IFN-gamma in 7 patients before vaccination and in 11 patients after vaccination. Of the latter 11 patients, 5 had detectable T cells both before and during the vaccination period, 4 only during the vaccination period, while 2 patients could for technical reasons not be assessed prevaccination. No induction of anti-PSA IgG or IgM antibodies was detected. There were no serious adverse events or otherwise severe toxicities observed during the trial. Immunization with Dendritophage-rPSA was feasible and safe in this cohort of patients. An immune response specific for PSA could be detected in some patients. A notable effect was the disappearance of circulating prostate cells in all patients who were RT-PCR positive before vaccination.