In 12 anesthetized dogs, we tested the effects of pharmacologic beta-adrenergic stimulation on the severity of tracheal mast cell reactions in vivo (Am Rev Respir Dis 1979; 119:62). In control dogs...

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcϵRI on mast cells and basophils by cross-linking FcϵRI with the inhibitory receptor FcγRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.

PROBLEM TO BE SOLVED: To provide an input specifying mechanism for efficiently supporting an auction and a dealing to maximize the profit of a seller while minimizing the cost of a buyer. SOLUTION: This method of processing the dealing comprises the steps of providing a solver/analyzer 42 determining one of an unrealizable distribution, a winning distribution and a realizable distribution, receiving a bid by the solver/analyzer 42, each bid having a bid price related to an item, relating the bid to dealing describing data, the dealing describing data having minimum/maximum liquidation values, free disposal, non-price attribute, adjustment, purpose, restriction, acquisition of realizability, relaxation of restriction, determination of conditional price, and features selected from estimate requirements, receiving the dealing describing data by the solver/analyzer 42, and processing the bid according to the features included in the dealing describing data. COPYRIGHT: (C)2004,JPO

It has long been recognized that IgE-mediated secretion from tissue mast cells or circulating basophils is a central pathophysiological mechanism responsible for the signs and symptoms of allergic diseases. It has also been recognized that elimination of IgE antibody from the circulation should ameliorate the pathophysiological process associated with atopy. Of course, not all aspects of allergic diseases have been shown to be fully dependent on IgE-mediated function in mast cells and basophils, but even in the case of asthma, where a wide variety of pathophysiological mechanisms probably play a role in the expression of the disease, eliminating the IgE-mediated component should have marked effects on the symptoms and severity of the disease. The trick has been developing a therapeutic that reduces circulating IgE and then providing enough of the agent to chronically and markedly influence the functional responses of IgE-bearing cells. This is not an easy task because these cells are exquisitely sensitive to stimulation. A great deal of knowledge about basophil and mast cell function can be used to make predictions about the requirements for new IgE-suppressing therapeutics. Such therapeutics, if actually demonstrated to suppress circulating IgE and associated cellular responses, could be useful tools for dissecting out the relative roles 40of this arm of the immune response in a variety of atopic diseases, including asthma. A variety of schemes have been proposed to induce a decrease in circulating IgE; one currently undergoing investigation is monoclonal anti-IgE antibody. Coupled with recent knowledge about basophil and mast cell function, studies using anti-IgE antibody in vivo have provided useful insights into the nature of allergic reactions, the cell biology of the high-affinity receptor for IgE (FcεRI), and their relationship to studies of basophil and mast cell function.

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human FcgRI, FcgRIIA, FcgRIIB, FcgRIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all FcgR; FcgRII and FcgRIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to FcgRIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/FcgRIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267–273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Immunoglobulin (Ig) E antibodies mediate allergic responses by binding to specific high affinity receptors, Fc epsilon RI, on mast cells and basophils. Previous studies have shown that the principal Fc epsilon RI binding site is located on the third constant domain, Fc epsilon 3, of IgE. Based on a model of the IgE Fc epsilon 3 (which is homologous to the second constant domain of IgG), homology scanning mutagenesis and replacement of individual residues were used to determine the specific amino acids of human IgE involved in binding to human Fc epsilon RI. The amino acids are localized in three loops, which form a putative ridge on the most exposed side of the Fc epsilon 3 domain of IgE and include Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469. The preponderance of charged residues suggests that IgE-Fc epsilon RI binding is mediated primarily by electrostatic interaction. Furthermore, it is possible to confer Fc epsilon RI binding to an IgG molecule by introducing these three IgE loops into the IgG C gamma 2 domain.

High IgE responder BDF1 mice were immunized intraperitoneally (i.p.) with dinitrophenol4 (DNP4)-ovalbumin (OVA) in alum concomitant with intravenous (i.v.) administration of an anti-IgE monoclonal antibody (mAb). IgE levels were undetectable in mice treated with the anti-IgE antibody, whereas mice treated with isotype-matched irrelevant mAb had IgE levels comparable to that of untreated, immunized mice. Subsequent antigen challenges with DNP4-OVA, either at weekly or monthly intervals, failed to evoke an IgE response for greater than 2 months in mice treated with anti-IgE during the primary sensitization, even though the terminal half-life of the anti-IgE antibody was 7 days. This inhibition was specific for DNP4-OVA since the DNP4-OVA-suppressed mice were able to respond to keyhole limpet haemocyanin (KLH). To investigate the effects of antibody treatment at the cellular level, passive transfer experiments were performed. The primary DNP-specific IgE response of adoptive transfer recipient mice was the same whether the donor cells were from mice treated with IgG or anti-IgE. Transfer of enriched T- or B-cell populations indicated that T-cell help was not compromised by administration of the anti-IgE mAb. However, splenocytes from the anti-IgE-treated mice failed to synthesize IgE in vitro, and flow cytometric analysis of B cells from anti-IgE-treated mice showed a dose-dependent decrease in CD23+ cells following antibody treatment, which correlated with decreased serum IgE levels. Taken together, the results of these studies suggest that anti-IgE treatment suppresses IgE responses via effects on B cells rather than T cells, possibly through effects on CD23-dependent pathways.