Eur. J. Immunol.

Endowing tumor cells with costimulatory signals for T cell activation has emerged as a promising strategy for tumor immunotherapy. Costimulatory molecules were either transfected into tumor cells to generate vaccines or were fused, e.g. to antibodies against tumor-associated antigens, to achieve targeted T cell costimulation in vivo. Here we report the production and purification of rM28, a recombinant bispecific single-chain antibody directed to a melanoma-associated proteoglycan and to the costimulatory CD28 molecule on human T cells. We found that a dimer of the recombinant molecule, bound to tumor target cells, induced pronounced T cell activation in peripheral blood mononuclear cell preparations without additional TCR/CD3 stimulation being required. The lytic activity generated after 3 days of stimulation effectively prevented tumor cell growth. However, it was unspecific and predominantly mediated by non T cells. Our findings demonstrate that presentation of a CD28 antibody within a suitable recombinant, bispecific format may result in a "targeted supra- agonistic stimulation" of the CD28 molecule, which leads to effective tumor cell killing after induction of unspecifically lytic cells

Supraagonistic, bispecific single-chain antibody purified from the serum of cloned, transgenic cows induces T-cell mediated killing of glioblastoma cells in vitro and in vivo

Here we charaeterize the antitumor activity of a reconbinant bispecific single-chain antibody isolated from the serurn of cloned transgenic cows. The antibody, termed r28M, is directed to a melanoma-associated proteogiycan, aiso expressed on gliobiastoma cells, and to human CD28. Bound to tumor cells, r28M induced exceedingiy efficient supraagonistic T-cell activation via the CD28 molecule without an additional stirnulus via the TCR/CD3 complex. In vitro, T cells and NK cells conlributed to tumor cell killing after r28M-mediated activation of peripheral biood mononuclear cells. However, NK activity depended on T-cell-derived cytokines. In vivo, r28M markedly inhibited the growth of human giioblastoma cells in nude mice. The serum haif-iife of the protein after i.v. injection was approximately 6 hr. Thus, r28M Is unique not oniy in inducing supraagonistic CD28-mediated T-cell activation against tumor cells in vitro and in vivo, it aiso meets 2 additional requirements that have critical for ciinical application: a relatively long serum half-life and the possibility of obtaining large amounts of active material from cloned transgenic livestock

Stimulus dependence of the action of small-molecule inhibitors in the CD3/CD28 signalling network.

Contains fulltext : 70946.pdf (publisher's version ) (Closed access)Cells in the body are exposed simultaneously to a multitude of various signals. Inside a cell, molecular signalling networks integrate this information into a physiologically meaningful response. Interestingly, in the cellular testing of drug candidates, this complexity is largely ignored. Compounds are tested for cells that are challenged with one stimulus only. The activation of T lymphocytes through engagement of the T cell receptor (TCR)-CD3 complex and CD28 coreceptor is a prominent example for a cellular response that depends on the integration of signals. We investigated the cellular response characteristics of this network at different strengths of receptor and coreceptor activation. A novel cellular microarray-based approach, in which various combinations of antibodies directed against the CD3 complex and CD28 were spotted, was employed for analysing the stimulus dependence of activation of the transcription factor NFAT and actin reorganisation. For both responses, quantitative differences in inhibitor activity were observed. Remarkably, for IL-2 expression, which was detected by standard ELISA, low doses of the Src-family kinase inhibitor PP2 strongly potentiated IL-2 expression at high-level, but not at low-level, CD28 co-engagement. Therefore, for a physiologically highly relevant signalling network, the cellular response might vary qualitatively with only quantitative variations of a stimulus. This level of complexity should be considered in early cellular drug testing

RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity

Cloned-transgenic farm animals produce a bispecific antibody for T-cell mediated tumor cell killing

Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being deveioped to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stabiiity, e.g., of singie-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yieid mg/ml levels of recombinant antibodies and the scale-up fiexibility make transgenic production in plants and iivestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific singie-chain antibody in the serum of transgenic rabbits arid a herd of nine cioned, transgenic cattie. The bispecific protein, designated r28M, Is directed to a meianoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animais, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing. scFv nuclear transfer DNA microinjection I antimelanoma anti-CD2