Recent progress in the understanding of immune function indicates that the interaction of CD40L with its receptor, CD40, plays a pivotal role in both humoral immunity and cell-mediated defense against pathogens. Functional studies of this interaction on both dendritic cells and malignant cells have demonstrated that CD40L also plays an important role in immune surveillance and anti-tumor immunity. CD40L exists in nature predominantly as a membrane-anchored molecule. To develop CD40L as a potential therapeutic, it is important to optimize soluble forms of this molecule that could be used in a clinical setting. Several reports have shown that soluble forms of CD40L, like CD40 antibodies, are biologically active. In the present report we demonstrate that the incorporation of an isoleucine zipper trimerization motif significantly enhances the biological activity of soluble CD40L.Interaction of CD40L (CD154) with its receptor, CD40, provides essential signals for the development of a protective humoral immune response and is a key mechanism in the regulation of defense against pathogenic assault (for review see Ref. 1). CD40, a 50-kDa transmembrane glycoprotein, is a member of the tumor necrosis factor (TNF) 1 receptor superfamily (for review see Refs. 2 and 3) and is expressed on antigen-presenting cells including dendritic cells, B cells, macrophages, follicular dendritic cells, and fibroblasts. CD40 is also expressed on a number of other cell types including endothelial cells and a significant proportion of carcinomas.CD40L is predominantly expressed on activated CD4 ϩ T cells, but variable expression has also been reported on CD8 ϩ T cells, mast cells, basophils, B cells, monocytes, NK cells, and activated platelets (1-4). Generally, CD40L does not exist naturally in a soluble form, although exceptions have been reported (5, 6). Initial studies focusing on the interaction of CD40L with its receptor on B cells show that CD40L is largely responsible for the helper T cell function that drives B cell proliferation and Ig class switching and protects B cells from apoptotic cell death. More recent studies on other cell types have indicated an important role for CD40L in the induction of T cell-mediated effector functions, antigen presentation, and costimulatory activity of antigen-presenting cells and in the production of many cytokines (for review see Ref. 1).CD40L is a type II membrane glycoprotein and is a member of the TNF superfamily. CD40L has an extracellular domain consisting of a 75-amino acid spacer region immediately adjacent to the membrane spanning region that is not shared by other family members and a receptor-binding domain that consists of two stacked ␤-sheets. The receptor-binding domain has low amino acid sequence homology with TNF family members (Ͻ30%) but has high structural homology (7). Soluble forms of CD40L have been expressed that are able to induce B cell proliferation, costimulate Ig class switching, and suppress the induction of apoptosis (8 -10). As with cell surface expressed CD40L, maxi...

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We have studied the cytotoxic effects of recombinant tumour necrosis factor and recombinant gamma interferon on primary cultures of leukaemia cells. The agents were added alone or in a combination to cells from 17 patients. Eleven had acute myeloblastic leukaemia (6 at presentation, 5 at relapse), 4 had acute lymphoblastic leukaemia, one had hairy cell leukaemia, and 2 had chronic myeloid leukaemia--one of whom was in myeloid blast transformation. Cells from patients with lymphoid malignancies or from the patient with chronic phase CML were not affected by either agent in any dose combination. In contrast, reduction of viability of myeloid blasts was weakly accelerated by TNF and gamma-interferon individually. Combination of the agents invariably produced enhanced killing and additive or synergistic effects were seen when 20-500 IU ml-1 of each cytokine was present. This sensitivity was also shown by blast cells from 5 patients with relapsed AML. We therefore suggest that trials of such combination therapy may be indicated in drug resistant or relapsed AML.

Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.