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.

Bispecific antibodies for polyclonal T-cell engagement.

Single-chain bispecific antibodies are emerging as an extremely powerful and promising class of polyclonal T-cell-engaging proteins with hitherto unknown properties. Such bispecific antibodies have the potential to bypass most T-cell escape mechanisms selected in tumor cells during their development into solid tumors and metastases. With cytotoxic T-cells, bispecific antibodies can recruit the most potent effector cells for tumor cell elimination. Studying the mechanism of action of newly developed single-chain bispecific antibodies could provide insight into the shortcomings of bispecific antibodies that have been studied previously.

Mode of cytotoxic action of T cell-engaging BiTE antibody MT110.

MT110 is an EpCAM/CD3-bispecific antibody construct in clinical development for the treatment of patients with adenocarcinoma expressing EpCAM (CD326). Like other members of this antibody class, MT110 can engage resting, polyclonal CD8(+) and CD4(+) T cells for highly potent redirected lysis of target cells. Here we further explored the mechanism of this action. Complete lysis of EpCAM(+) Kato III gastric cancer cells by previously unstimulated T cells was achieved within 48 h. During this period, a high percentage of CD4(+) and CD8(+) T cells became activated and increased expression of granzyme B. This apparently boosted the capacity for serial target cell lysis as studied at very low effector-to-target ratios. Elimination of cancer cells by MT110-redirected T cells involved membrane damage as was evident from nuclear uptake of propidium iodide and release of the cytosolic enzyme adenylate kinase. Redirected T cells also potently triggered programmed cell death in cancer cells as was evident by membrane blebbing, activation of procaspases 3 and 7, fragmentation of nuclear DNA and cleavage of the caspase substrate poly (ADP ribose) polymerase. Chelation of extracellular calcium fully protected cancer cells from lysis by MT110-redirected T cells, while the pan-caspase inhibitor Z-VAD-FMK blocked activation of procaspases, cleavage of poly (ADP ribose) polymerase and fragmentation of nuclear DNA in cancer cells, but could not prevent nuclear uptake of propidium iodide. Soluble factors did not significantly contribute to cancer cell death. Our study shows that MT110 can efficiently gear up the potential of CD8(+) and CD4(+) T cells for serial lysis, and mediate kill of cancer cells predominantly through poreforming and pro-apoptotic components of cytotoxic T cell granules.

Potent inhibition of local and disseminated tumor growth in immunocompetent mouse models by a bispecific antibody construct specific for Murine CD3.

Bispecific single-chain antibody constructs specific for human CD3 have been extensively studied for antitumor activity in human xenograft models using severe combined immunodeficient mice supplemented with human T cells. High efficacy at low effector-to-target ratios, independence of T cell costimuli and a potent activation of previously unstimulated polyclonal T cells were identified as hallmarks of this class of bispecific antibodies. Here we studied a bispecific single-chain antibody construct (referred to as 'bispecific T cell engager', BiTE) in an immunocompetent mouse model. This was possible by the use of a murine CD3-specific BiTE, and a syngeneic melanoma cell line (B16F10) expressing the human Ep-CAM target. The murine CD3-specific BiTE, called 2C11x4-7 prevented in a dose-dependent fashion the outgrowth of subcutaneously growing B16/Ep-CAM tumors with daily i.v. injections of 5 or 50 microg BiTE which was most effective. Treatment with 2C11x4-7 was effective even when it was started 10 days after tumor cell inoculation but delayed treatments showed a reduction in the number of cured animals. 2C11x4-7 was also highly active in a lung tumor colony model. When treatment was started on the day of intravenous tumor cell injection, seven out of eight animals stayed free of lung tumors, and three out of eight animals when treatment was started on day 5. Our study shows that BiTEs also have a high antitumor activity in immunocompetent mice and that there is no obvious need for costimulation of T cells by secondary agents.

Highly efficient antigen targeting to M-DC8+ dendritic cells via FcgammaRIII/CD16-specific antibody conjugates.

Conjugates of peptide antigens with antibodies specifically recognizing surface molecules on dendritic cells (DC) represent an attractive approach to target antigens to antigen-presenting cells (APC) for the induction of specific T cell responses. The present study evaluates the potential of M-DC8(+) DC, a sub-population of professional APC in the blood, for an antibody-based vaccination strategy. We prepared, by chemical cross-linking, conjugates of peptide model antigens with antibodies directed against different cell surface molecules of DC. Antigen-peptide conjugates using an anti-CD16 (FcgammaRIII) antibody were most potent in inducing in vitro activation of a specific CD4(+) T cell response. They were at least 300 times more efficient than two other antibody-antigen conjugates and approximately 500 times more efficient than unconjugated antigen peptides. Our data demonstrate that specific antigen targeting via CD16 on M-DC8(+) DC is a promising vaccination approach for the efficient induction of specific CD4(+) T cell responses ex vivo, and perhaps in vivo.

The promise of bispecific antibodies.

Extract: While monoclonal antibodies closely resemble the naturally occurring immune defense proteins, bispecific antibodies (i.e., antibodies detecting two different antigens) are not found in nature. Both types of antibodies share the feature of antigen specificity that is mediated by the presence of a unique antigen binding site on the antibody. Antigen binding sites exclusively interact with a particular molecular part of an antigen, known as an epitope. Monoclonal IgG (immunoglobulin G) antibodies carry two antigen binding sites for the same epitope, while bispecific antibodies are equipped with two different antigen binding sites with the potential to link two epitopes that could be present on two different cell types. Murine monoclonal antibodies first became available through the hybridoma technology developed by Köhler and Milstein in 1975. Since then they have been improved for therapeutic use by chimerization (only the antigen binding sites remain murine, all other antibody parts are of human origin, e.g., Erbitux), humanization (only the six subregions of the antigen binding site that actually contact the epitope remain murine, e.g., Avastin) or the recovery of fully human antibodies from phage display libraries or from mice transgenic for human immunoglobulin genes (e.g., ABX-EGF, an investigational drug). Several monoclonal antibodies have been approved for human therapy in various disease indications acting either as receptor antagonists, neutralizers of protein receptor ligands or through the recruitment of immune effector functions against pathogenic cells.

T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct.

We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.