MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors.

We have developed a novel single-chain Ep-CAM-/CD3-bispecific single-chain antibody construct designated MT110. MT110 redirected unstimulated human peripheral T cells to induce the specific lysis of every Ep-CAM-expressing tumor cell line tested. MT110 induced a costimulation independent polyclonal activation of CD4- and CD8-positive T cells as seen by de novo expression of CD69 and CD25, and secretion of interferon gamma, tumor necrosis factor alpha, and interleukins 2, 4 and 10. CD8-positive T cells made the major contribution to redirected tumor cell lysis by MT110. With a delay, CD4-positive cells could also contribute presumably as consequence of a dramatic upregulation of granzyme B expression. MT110 was highly efficacious in a NOD/SCID mouse model with subcutaneously growing SW480 human colon cancer cells. Five daily doses of 1 microg MT110 on days 0-4 completely prevented tumor outgrowth in all mice treated. The bispecific antibody construct also led to a durable eradication of established tumors in all mice treated with 1 microg doses of MT110 on days 8-12 after tumor inoculation. Finally, MT110 could eradicate patient-derived metastatic ovarian cancer tissue growing under the skin of NOD/SCID mice. MT110 appears as an attractive bispecific antibody candidate for treatment of human Ep-CAM-overexpressing carcinomas.

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.

Concerted antigen presentation by dendritic cells and B cells is necessary for optimal CD4 T-cell immunity in vivo.

The relative contributions of different types of antigen presenting cells to T-cell activation, expansion and induction of effector functions are still not fully understood. In order to evaluate the roles of dendritic versus B cells during these phases of a CD4 T-cell response in vivo, we adoptively transferred major histocompatibility complex class II restricted, T-cell receptor-transgenic CD4+ T cells into transgenic mice expressing selectively the T-cell restricting class II molecules on either dendritic cells, B cells or both. Upon immunization with peptide antigen, we observed that dendritic cells were sufficient to induce activation, expansion, interleukin-2 production and germinal centre migration of antigen-specific T cells, independently of other antigen-presenting cells. In contrast, neither resting nor activated B cells had similar antigen-presenting capacities in vivo. However, in double transgenic mice where both B cells and dendritic cells were capable of presenting antigen, T cells showed increased proliferation, expansion and cytokine production in vivo. Moreover, higher antigen-specific CD4 T-cell numbers accumulated in germinal centres. Our data demonstrate that dendritic cells are sufficient to activate naive CD4 T cells in vivo, but B cells subsequently can enhance CD4 T-cell expansion further.

Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct.

Bispecific T-cell engager (BiTE) are a class of bispecific single-chain antibodies that can very effectively redirect cytotoxic T cells for killing of tumor target cells. Here, we have assessed the in vivo efficacy of one representative, called bscEp-CAMxCD3, with specificity for tumors overexpressing epithelial cell adhesion molecule (Ep-CAM) in human xenograft models. Cells of the human colon carcinoma line SW480 were mixed at a 1:1 ratio with unstimulated human peripheral mononuclear cells, s.c. injected in nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, and animals were treated with bscEp-CAMxCD3. Five daily i.v. injections of as little as 100 ng per mouse of bscEp-CAMxCD3 completely prevented tumor outgrowth when treatment was started at the day of tumor cell inoculation. BscEp-CAMxCD3 was also efficacious when administered up to 8 days after xenograft injection. Established tumors could be eradicated in all animals by five 10 microg doses given between days 8 and 12 after tumor cell inoculation. To test the efficacy of bscEp-CAMxCD3 in a more physiologic model, pieces of primary metastatic tumor tissue from ovarian cancer patients were implanted in NOD/SCID mice. Partial tumor engraftment and growth was observed with four of six patient samples. Treatment of established tumors with daily 5 microg doses led to a significant reduction and, in some cases, eradication of human tumor tissue. These effects obviously relied on the tumor-resident T cells reactivated by bscEp-CAMxCD3. Our data show that the class of single-chain bispecific antibodies has very high antitumor efficacy in vivo and can use previously unstimulated T cells at low effector-to-target ratios.

Simultaneous induction of CD4 T cell tolerance and CD8 T cell immunity by semimature dendritic cells.

Previous studies suggested that depending on their maturation state, dendritic cells (DC) could either induce T cell tolerance (immature and semimature DC) or T cell activation (mature DC). Pretreatment of C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide-loaded semimature DC protected from MOG-induced autoimmune encephalomyelitis. This protection was mediated by IL-10-producing CD4 T cells specific for the self Ag. Here we show that semimature DC loaded with the MHC class II-restricted nonself peptide Ag (OVA) induce an identical regulatory T cell cytokine pattern. However, semimature DC loaded simultaneously with MHC class II- and MHC class I-restricted peptides, could efficiently initiate CD8 T cell responses leading to autoimmune diabetes in a TCR-transgenic adoptive transfer model. Double-peptide-loaded semimature DC also induced simultaneously in the same animal partially activated CD8 T cells with cytolytic function as well as protection from MOG-induced autoimmune encephalomyelitis. Our study suggests that the decision between tolerance and immunity not only depends on the DC, but also on the type and activation requirements of the responding T cell.

Endogenous dendritic cells are required for amplification of T cell responses induced by dendritic cell vaccines in vivo.

Dendritic cells (DCs) loaded in vitro with Ag are used as cellular vaccines to induce Ag-specific immunity. These cells are thought to be responsible for direct stimulation of Ag-specific T cells, which may subsequently mediate immunity. In this study, in transgenic mouse models with targeted MHC class II expression specifically on DCs, we show that the DC vaccine is responsible only for partial CD4(+) T cell activation, but to obtain optimal expansion of T cells in vivo, participation of endogenous (resident) DCs, but not endogenous B cells, is crucial. Transfer of Ag to endogenous DCs seems not to be mediated by simple peptide diffusion, but rather by DC-DC interaction in lymph nodes as demonstrated by histological analysis. In contrast, injection of apoptotic or necrotic DC vaccines does not induce T cell responses, but rather represents an immunological null event, which argues that viability of DC vaccines can be crucial for initial triggering of T cells. We propose that viable DCs from the DC vaccine must migrate to the draining lymph nodes and initiate a T cell response, which thereafter requires endogenous DCs that present transferred Ag in order induce optimal T cell expansion. These results are of specific importance with regard to the applicability of DC vaccinations in tumor patients, where the function of endogenous DCs is suppressed by either tumors or chemotherapy.