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Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMCs) derived from healthy donors were cocultured with melanoma cells at effector:target ratios of 1:1, 1:5, or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100, or 1000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose-dependent and effector:target ratio-dependent manner, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we cocultured unstimulated PBMCs with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8 T cells were prestimulated by anti-CD3 antibody OKT-3 and interleukin-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. As MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.

Inhibition of the interleukin-2 (IL-2) pathway has potent immunosuppressive activity in humans as is evident from the broad therapeutic utility of cyclosporine, rapamycin, tacrolimus, and monoclonal antibodies blocking the high-affinity subunit of the IL-2 receptor (CD25). Here we describe a humanized antibody, MT204, interfering with IL-2 signaling by a novel mechanism. Although MT204 did not prevent IL-2 from binding to CD25, it potently antagonized downstream signaling events of IL-2 at sub-nanomolar concentrations, such as STAT3 tyrosine phosphorylation, expression of CD124, production of gamma-interferon and cell proliferation. While MT204 and the anti-CD25 mAb daclizumab were equally effective in inhibiting autocrine growth of human CD4(+) T cells, MT204 was far superior in preventing proliferation of NKL lymphoma cells, production of gamma-interferon by natural killer (NK) cells and proliferation of primary NK cells. MT204 has potential as a novel immunosuppressive and anti-proliferative therapy with an apparently broader spectrum of activities than anti-CD25 antibodies.

The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably “gain of function” to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.

Synthetic oligodeoxynucleotides containing CpG dinucleotides (CpG-ODN) mimic the immunostimulatory qualities of bacterial DNA. We asked whether immunostimulation by CpG-ODN predisposes for a commitment toward a Th1 vs a Th2 response in Leishmania major infection, a model for a lethal Th2-driven disease, in BALB/c mice. CpG-ODN induced Th1 effector T cells in vitro and conveyed protective immunity to disease-prone BALB/c mice in vivo. Conversion to a Th1-driven resistant phenotype was associated with IL-12 production and maintained the expression of IL-12R β2-chains. Most strikingly, CpG-ODN were even curative when given as late as 20 days after lethal L. major infection, indicating that CpG-ODN revert an established Th2 response. These findings imply an important role of bacterial DNA and CpG-ODN in the instruction of adaptive immune responses. They also point to the therapeutic potential of CpG-ODN in redirecting curative Th1 responses in Th2-driven disorders.

One of the main components of WHO’s current malaria control strategy centres on early recognition and prompt treatment. Rapid identification of malaria and adequate treatment are essential for preventing irreversible complications and most deaths can be avoided. But even in areas with good access to health care, it is common for malaria patients to present at the health facility late or not at all. The overall goal of the study was to investigate community understanding of malaria, and treatment-seeking, and the way in which this can contribute to delay in attending a health facility in order to formulate recommendations for tackling this problem. The study was set in Ifakara (Kilombero District) in southeastern Tanzania, a semi-rural town with a large and well equipped District Hospital and other, private and public health facilities and pharmacies. Antimalarials, mainly chloroquine and other drugs are also widely available over-the-counter in the numerous small shops throughout the community. Beside the biomedical resources, traditional medicine is well represented in the community. Many different types of traditional healers offer their services to the public. Malaria in the study area is holoendemic and perennial, which presents a huge health burden for the population, in particular to children under the age of five years. Resistance to chloroquine was found to be high. The ethnographic fieldwork was conducted in a two year field study carried out between April 1995 and March 1997. Additional information has been recorded in a second, short field visit of one month in September 1997 in the context of a study on the impact of cost-sharing on the community, requested by the St. Francis Designated District Hospital. The investigation strategy was that of triangulation, using a combination of qualitative and quantitative methods. The population was found to be very well informed about malaria as it is biomedically defined. However, one of the major findings was that the local knowledge is the result of an interplay between biomedical and traditional concepts and logics, a process which is referred to as ‘medical syncretism’. Analysing the amalgamation of the two types of knowledge contributed to the understanding of cultural logics underlying treatment-seeking behaviour for malaria. The study of medical syncretism reveals most clearly that even if health messages are well understood by the population, the meaning given to them may considerably differ from what health promoters intended to convey. This amalgamation and its consequences for treatment and delay was seen as relevant for all three forms of malaria (uncomplicated, severe, and recurrent malaria). For example, in the case of uncomplicated malaria, it was found that cultural logics derived from notions on witchcraft could lead people to misinterpret repeated vomiting as a sign of relief, rather than as a manifestation indicating an evolution from uncomplicated to severe malaria. For severe malaria, the local illness term degedege was identified to come closest to biomedically defined cerebral malaria. However, while informants clearly recognised the link between degedege and malaria, they did not treat the two forms in the same way; for degedege, people used primarily traditional practices, including assistance by ‘knowledgeable women, while for malaria, they preferred biomedical treatments by far. Based on this finding, the role of knowledge about aetiology for treatment-seeking is discussed. It is argued that knowledge which is present in the cultural repertoire (‘recipe knowledge’) and is automatically evoked for action (non-reasoned action) plays an important role in treatment-seeking, especially for treatments in an early phase of illness. Reasoned action was found to set in when something unexpected occurs, for example when symptoms inexplicably aggravate or persist despite treatment. The study showed how in the local illness model, biomedical ideas about malaria are complemented with the logics of witchcraft. The analysis of the resulting knowledge permitted us to explain the logics which guide people in their labyrinthic treatment-seeking paths, including biomedical and traditional health services for the same illness episode. In a second focus, the study emphasised the relevance of economic constraints for treatment-seeking. Cognitive aspects were linked with social and economic aspects. It was observed that perception about illness aetiology determined the implication of the social network for illness management and the social pressure on covering treatment costs of the sick individual. In contrast to illnesses attributed to witchcraft or spirits, for illnesses which belonged to the ‘natural’ order, such as malaria, and required hospital intervention, support networks for coping viiwith treatment costs, if any, were found to be small. Women who could not count on male support were identified to be at a particular risk for delaying treatment for their children because of economic reasons. It was found that women had adopted different coping strategies for covering treatment costs, but besides delay, they frequently had negative long-term implications for their and their children’s well-being. A particular high risk for delay was found to result from a negative interaction of gender, seasonality and illness factors. The findings from this study contributed to a rethinking of the traditional - modern dichotomy in socio-cultural malaria research. They further challenged the common view that traditional treatments are an important source of delay for malaria treatments and called for the need to increasingly focus research on delay and application on socio-economic perspectives. Direct implications of the study for further research and recommendations for action were extensively discussed.

BiTE antibodies are designed to transiently connect target cells with T cells of patients for induction of potent redirected lysis of cancer cells. Clinical proof-of-concept has been obtained with CD19/CD3-bispecific BiTE antibody blinatumomab (MT103), which showed a high response rate in patients with relapsed/refractory non-Hodgkin's lymphoma and acute B-lymphocytic leukemia. An EpCAM/CD3-bispecific BiTE antibody called MT110 currently is in a dose-escalating phase 1 study in patients with lung or gastrointestinal cancers, and BiTE antibodies targeting carcinoembryonal antigen (CEA), EGFR, CD33 or melanoma chondroitin sulfate proteoglycan (MCSP) are in earlier stages of development. Here, we have generated a series of novel BiTE antibodies for cancer therapy that are based on our novel BiTE antibody platform, which is human in sequence and cross-reactive between human and non-human primate antigens. One set of target antigens selected for new BiTE generation is also targeted by conventional monoclonal antibodies that are in various stages of clinical development. These are mesenchymal-epithelial transition factor (cMet), insulin-like growth factor receptor type I (IGFR-1), prostate-specific stem cell antigen (PSCA), carboanhydrase type IX (CAIX) and Her2/neu. These targets are all expressed by cancer cells of solid tumors, and some also by their so-called cancer stem cells. By targeting fibroblast activating protein alpha (FAP-alpha), a first BiTE antibody was generated that not only targets sarcoma cells but also stromal fibroblasts, which constitute the stromal tissue of many human cancers. By targeting endosialin (CD248), a first BiTE antibody was generated targeting cancer blood vasculature. We show that various single-chain antibodies specific for the seven target antigens cMet, IGFR-1, FAP-alpha, PSCA, CAIX, Her2/neu and endosialin could in each case be used to construct novel BiTE antibodies. All BiTE antibodies showed bispecific binding, cross-reactivity between human and Cynomolgus monkey antigens, and, most importantly, potent redirected lysis of CHO cells expressing the respective target antigen. This shows that new T cell-engaging BiTE antibodies can be made specific for a great variety of potential tumor-associated and also for stromal antigens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2434.

Melanoma chondroitin sulphate proteoglycan (MCSP; also called CSPG4, HMW-MAA, MSK16, MCSPG, MEL-CSPG, NG2, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signalling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. BiTE antibodies can transiently connect the T cell receptor subunit CD3 on T cells with a surface antigen on target cells leading to a highly efficient redirected lysis of target cells. This involves cytolytic synapse formation and delivery of perforin and granzymes. BiTE-engaged T cells are capable of serial target cell lysis, and are not affected by immune escape mechanisms interfering with peptide antigen processing and presentation, or clonal T cell differentiation. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO target cells stably transfected with full-length human MCSP, MCSP deletion mutants or fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by using MCSP/EpCAM fusion proteins as surface target antigens. Generally, CHO cells expressing small surface antigens were better lysed than those with large antigens, indicating that antigen size is also an important factor for BiTE potency. This is in line with the observation that target cell lysis by membrane proximally-binding BiTE antibodies improved upon omission of more distal MCSP domains. The present study for the first time relates the size of surface antigens and positioning of binding domains to BiTE potency of target cell lysis via redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5340.