Combination of rituximab with blinatumomab (MT103/MEDI-538), a T cell-engaging CD19-/CD3-bispecific antibody, for highly efficient lysis of human B lymphoma cells.

We have compared the cytotoxic activity of rituximab with that of blinatumomab (MT103/MEDI-538), a single-chain CD19-/CD3-bispecific antibody engaging human T cells. Blinatumomab consistently led to a higher degree of lysis of human lymphoma lines than rituximab, and was active at much lower concentration. The cytotoxicity mediated by blinatumomab and rituximab both caused a potent activation of pro-caspases 3 and 7 in target cells, a key event in induction of granzyme-mediated apoptotic cell death. Combination of rituximab with blinatumomab was found to greatly enhance the activity of rituximab, in particular at low effector-to-target cell ratios and at low antibody concentration.

A multimarker real-time RT-PCR for MAGE-A gene expression allows sensitive detection and quantification of the minimal systemic tumor load in patients with localized cancer.

INTRODUCTION: Distant metastases of solid tumors are usually associated with fatal outcome. Disseminated cancer cells are considered early indicators of metastasis. Their sensitive detection and quantification would be a valuable tool for staging of disease and as guidance for therapeutic decisions. EXPERIMENTAL DESIGN: We established a highly sensitive and quantitative multimarker real-time RT-PCR assay for amplification of cancer-related genes MAGE-A1, -A2, -A3/6, -A4, -A10 and -A12 using SYBR green I to detect one single tumor cell in 2 mL of blood or bone marrow. The feasibility of the assay was tested in a large cohort of 177 patients with locally confined prostate carcinoma. RESULTS: Analysis revealed frequent MAGE expression in venous blood and bilateral bone marrow samples (25.5% of all cases) and yielded the first quantitative profile of MAGE expression with a broad range of transcript concentrations for individual markers in the minimal systemic tumor load of patients with localized cancer. CONCLUSIONS: Rare transcripts of different MAGE-A genes can be quantified in clinical samples of cancer patients by a sensitive multimarker real-time RT-PCR. Because of frequent expression of MAGE genes in various types of cancer the multimarker MAGE real-time RT-PCR may be generally useful for detection, quantification and characterization of the individual disseminated tumor load in cancer patients.

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct.

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3x10(-7) M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.

A phase I study with adecatumumab, a human antibody directed against epithelial cell adhesion molecule, in hormone refractory prostate cancer patients.

AIM OF THE STUDY: Adecatumumab (also known as MT201) is a human recombinant IgG1 monoclonal antibody binding with low affinity to epithelial cell adhesion molecule (EpCAM). To explore safety, pharmacokinetics and pharmacodynamics of adecatumumab, a phase I trial in patients with hormone refractory prostate cancer (HRPC) was performed. METHODS: Twenty patients were treated with two adecatumumab infusions on days 0 and 14 in cohorts with doses of ten up to 262 mg/m2. RESULTS: Adecatumumab was well tolerated at all doses tested, and no maximum tolerated dose reached. Most adverse events were mild or moderate with pyrexia and nausea being most frequent. The highest dose of adecatumumab induced shortly after infusion robust and transient increases of TNF-alpha serum levels. At all doses, significant transient declines of peripheral natural killer cells were observed shortly after antibody infusions. Adecatumumab had a serum half-life of 15 days, and immune responses to the antibody were not detected. CONCLUSIONS: A benign safety profile, long serum half-life and low immunogenicity do warrant further exploration of adecatumumab for treatment of EpCAM-expressing neoplasia.

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct.

BscCD19xCD3 is a bispecific single-chain antibody construct with exceptional cytotoxic potency in vitro and in vivo. Here, we have investigated the biological activity of bscCD19xCD3 in chimpanzee, the only animal species identified in which bscCD19xCD3 showed bispecific binding, redirected B-cell lysis and cytokine production comparable to human cells. Pharmacokinetic analysis following 2-h intravenous infusion of 0.06, 0.1 or 0.12 mug/kg of bscCD19xCD3 as part of a dose escalation study in a single female chimpanzee revealed a half-life of approximately 2 h and elimination of the bispecific antibody from circulation within approximately 8 h after the end of infusion. This short exposure to bscCD19xCD3 elicited a transient increase in serum levels of IFNgamma, IL-6, IL-2, soluble CD25, and transiently upregulated expression of CD69 and MHC class II on CD8-positive cells. Cytokine release and upregulation of T-cell activation markers were not observed with vehicle controls. A multiple-dose study using 5 weekly doses of 0.1 mug/kg in two animals also showed transient cytokine release and an activation of peripheral T cells with a first-dose effect, accompanied by a transient lymphopenia. While oscillations of T-cell counts were relatively even during repeated treatments, the amplitudes of peripheral B cells declined with every infusion, which was not observed in a vehicle control animal. Our data show that bscCD19xCD3 can be safely administered to chimpanzees at dose levels that cause fully reversible T-cell activation and, despite a very short exposure time, cumulative loss of peripheral B lymphocytes. A clinical trial testing prolonged administration of bscCD19xCD3 (MT103) for improving efficacy is currently ongoing.

High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G.

A common feature of human IgG1 antibodies used for cancer treatment is that their anti-tumour efficacy requires high serum trough levels and continued therapy for several months. Treatment cycles, thereby, consume several grams of IgG1 translating into significant drug needs and costs. The basis for the low in vivo efficacy, which is in contrast to high in vitro antibody-dependent cellular cytotoxicity (ADCC), is not well understood. Here, we have explored factors contributing to this discrepancy using adecatumumab (MT201), a fully human monoclonal IgG1 against epithelial cell adhesion molecule (Ep-CAM) and trastuzumab (Herceptin), a humanized IgG1 with specificity for the human epithelial growth factor receptor type 2 (HER-2) antigen. We found that physiological levels of human sera strongly inhibited ADCC of both IgG1 antibodies. Effects showed some dependence on the density of Ep-CAM and HER-2 targets, the tumour cell line tested and on effector cell and serum donors. Removal of IgG by affinity chromatography abolished the inhibitory effect of a serum pool. Inhibition of ADCC was fully restored by adding back the IgG fraction or by an equal amount of IgG from a commercial source. We further demonstrate that CD56-positive lymphocytes within human PBMC contributed >90% to ADCC and that normal serum levels of IgG effectively competed for in vitro binding of an IgG1 antibody to low-affinity Fcgamma receptor type III (CD16), as is present on natural killer (NK) cells. Competition of serum IgG for binding of therapeutic IgG1 to NK cell may be one important reason why high antibody doses are required in the clinic for treatment of cancer by an ADCC-based mechanism.

Expression of epithelial cellular adhesion molecule (Ep-CAM) in chronic (necro-)inflammatory liver diseases and hepatocellular carcinoma.

Epithelial cell adhesion molecule (Ep-CAM) is expressed in a several epithelial tissues and carcinomas, but not on mature hepatocytes. Here, we analysed the expression of Ep-CAM in 230 patients suffering from various liver diseases like chronic hepatitis B and C (HBV and HCV infection), chronic autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hereditary hemochromatosis and dysplastic nodules (DNs) as well as hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs) by immunohistochemistry. De novo hepatocellular Ep-CAM expression was found in 75.9% of ALD (22/29), 63.6% of HCV (21/33) and 55.6% of each AIH and HBV cases (5/9 and 15/27, respectively). Lower Ep-CAM expression levels were observed for primary sclerosing liver diseases (PBC and PSC) with 25% (3/12) and 7.7% (1/13) of cases. Moreover, only 14.3% of HCCs (9/63) manifested expression, while all CCCs showed strong Ep-CAM expression (5/5). For DNs and hereditary hemochromatosis, Ep-CAM expression was found in 10 and 50% (3/30 and 2/4), respectively. In HBV and HCV, Ep-CAM expression correlated significantly with inflammatory activity as assessed by histological parameters and to the extent of fibrosis. In addition, for HCV also transaminase levels correlated significantly with Ep-CAM expression. Our results indicate that de novo Ep-CAM expression in hepatocytes is frequent in inflammatory liver diseases and is potentially linked to regenerative activity. CCCs and Ep-CAM positive HCCs may represent an attractive target group for Ep-CAM-directed immunotherapies, yet unwanted toxicity may limit the use of such strategies due to Ep-CAM expression in biliary epithelium and several chronic liver diseases such as HBV-and HCV-hepatitis.

Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct.

Certain bispecific antibodies exhibit an extraordinary potency and efficacy for target cell lysis by eliciting a polyclonal T-cell response. One example is a CD19-/CD3-bispecific single-chain antibody construct (bscCD19xCD3), which at femtomolar concentrations can redirect cytotoxic T cells to eliminate human B lymphocytes, B lymphoma cell lines and patient-derived malignant B cells. Here we have further explored the basis for this high potency. Using video-assisted microscopy, bscCD19xCD3 was found to alter the motility and activity of T cells from a scanning to a killing mode. Individual T cells could eliminate multiple target cells within a 9 hr time period, resulting in nuclear fragmentation and membrane blebbing of target cells. Complete target cell elimination was observed within 24 hr at effector-to-target cell ratios as low as 1:5. Under optimal conditions, cell killing started within minutes after addition of bscCD19xCD3, suggesting that the rate of serial killing was mostly determined by T-cell movement and target cell scanning and lysis. At all times, T cells remained highly motile, and no clusters of T and target cells were induced by the bispecific antibody. Bystanding target-negative cells were not detectably affected. Repeated target cell lysis by bscCD19xCD3-activated T cells increased the proportion of CD19/CD3 double-positive T cells, which was most likely a consequence of transfer of CD19 from B to T cells during cytolytic synapse formation. To our knowledge, this is the first study showing that a bispecific antibody can sustain multiple rounds of target cell lysis by T cells.

Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease.

Systemically disseminated tumor cells have become the subject of intensive research as the presumed seminal precursors of later distant metastasis. We describe here a novel sensitive multimarker nested reverse transcription (RT)-PCR capable of detecting the individual expression of human MAGE-A genes MAGE-1, -2, -3/6, -4, and -12 by rare, disseminated tumor cells in bone marrow and blood of patients with many different types of cancer. We analyzed bone marrow aspirates from 106 patients with breast, lung, colorectal, and prostate cancer and with different sarcomas. Heterogeneous expression of the different MAGE genes was found frequently in all those kinds of malignancies, in sharp contrast to 30 bone marrow and 20 blood samples from healthy donors, which were completely MAGE negative. Expression of at least one MAGE gene in bone marrow was more frequent than cytokeratin-positive tumor cells detected by immunocytochemistry, although the results of both tests overlapped considerably. In 30 patients with clinically localized prostate cancer, analysis by the multimarker MAGE RT-PCR of bilateral bone marrow aspirates from the right and left iliac crest revealed a positivity rate of 60%, which was twice as high as that obtained with either an established prostate-specific antigen RT-PCR or by cytokeratin-based immunocytochemistry. Analysis of primary prostate cancer revealed MAGE expression patterns considerably concordant with those found in the corresponding bone marrow aspirates. Prostate cancer patients carrying an exceptionally high risk of metastatic relapse, as defined by clinical prognostic factors, were significantly more often MAGE positive than patients with a distinctly lower risk (P = 0.02, Fisher's exact test). More frequent MAGE expression in the peripheral blood of patients with metastatic prostate cancer compared with those with clinically localized disease added further evidence for the prognostic impact of the multimarker MAGE RT-PCR. Moreover, MAGE-positive bone marrow samples from a small group of seven sarcoma patients demonstrated the relevance of our multimarker RT-PCR in nonepithelial tumors. Because MAGE antigens can induce autologous cytolytic T lymphocytes in vivo, the determination of individual MAGE expression patterns in cancer patients may furthermore identify candidate vaccine targets for adjuvant immunotherapy.