Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig.

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.

Arabinosylation of recombinant human immunoglobulin-based protein therapeutics.

Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor.

The TNF antagonists adalimumab, infliximab, and etanercept are effective treatments for rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and psoriasis, but only adalimumab and infliximab have been found to be efficacious in Crohn's disease. The present studies evaluated the TNF-binding and complement-activating properties of adalimumab, infliximab, and etanercept to determine whether these properties may explain differences in their clinical efficacy profiles. Association and dissociation rates of binding to soluble TNF were measured by surface plasmon resonance, and were found to be similar for adalimumab, infliximab, and etanercept, as were their calculated binding affinities. Avidity of binding to soluble TNF, measured by KinExA technology, was 10- to 20-fold greater for soluble etanercept (K(D)=0.4 picomolars [pM]) than for soluble adalimumab or infliximab (K(D)=8.6 and 4.2 pM, respectively). (125)I-adalimumab, -infliximab, and -etanercept bound to membrane TNF (mTNF) on mTNF-transfected cells with similar affinities (K(D)=483, 468, and 445 pM, respectively) that were each lower than for soluble TNF. Complement-dependent cytotoxicity (CDC) was induced in mTNF-transfected cells by adalimumab and infliximab, but was not induced in activated normal human PBMC by any of the 3 agents. In conclusion, the binding properties of adalimumab, infliximab, and etanercept were similar for soluble TNF, and very similar for mTNF, yet none of the 3 was able to induce CDC in activated PBMC. These results suggest that the different clinical efficacy profiles of these agents are not explained by differences in either TNF-intrinsic binding properties or complement lysis.

Synthesis and complement inhibitory activity of B/C/D-ring analogues of the fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy- 2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran].

This paper reports the synthesis and the bioassay of 4-methoxy- and 4-hydroxyspiro[benzofuran-2(3H)-cyclohexane] partial analogues (5) of the complement inhibitory sesquiterpene fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy-2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a, K-76) and its silver oxide oxidized product (1b, K-76COOH). The described target compounds represent spirobenzofuran B/C/D-ring analogues lacking the A-ring component of the prototype structure. The target compounds were evaluated by the inhibition of total hemolytic complement activity in human serum. It was observed that the structurally simplified analogue 4-methoxyspiro[benzofuran-2(3H)-cyclohexane]-6-carboxylic acid (5a) exhibited an IC(50) = 0.53 mM similar to the IC(50) = 0.57 mM that was observed for the natural product derivative 1b. Exhibiting an IC(50) = 0.16 mM, the three-ringed partial structure 6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (5k)was found to be the most potent target compound. Like the natural product, 5k appears to inhibit primarily at the C5 activation step and inhibits both the classical and alternative human complement pathways. Several other analogues inhibited complement activation in vitro at concentrations similar to those required for inhibition by the natural product 1b.

IL-18 receptor beta-induced changes in the presentation of IL-18 binding sites affect ligand binding and signal transduction.

IL-18 is a pleiotropic proinflammatory cytokine that is involved in induction of inflammatory mediators, regulation of the cytotoxic activity of NK cells and T cells, and differentiation and activation of both Th1 and Th2 cells. IL-18 signals through its specific cell surface receptor IL-18R, which comprises two subunits: IL-18R alpha and IL-18R beta. IL-18R alpha alone has a weak affinity for IL-18 binding, while the IL-18R alpha/beta complex has a high affinity. By using several anti-IL-18 mAbs and IL-18 binding protein, we have examined whether these site-specific inhibitors could block the binding of IL-18 to IL-18R alpha and to the IL-18R alpha/beta complex. Here we show that IL-18 binding to IL-18R alpha was inhibited by a neutralizing mAb, 125-2H, while binding of IL-18 to the alpha/beta receptor complex was not. This suggests that IL-18R beta-induced conformational changes may occur in IL-18R alpha upon dimerization, leading to changes in the presentation of IL-18 binding sites. Epitope mapping of 125-2H using human-mouse IL-18 chimeras identified a region in IL-18 that was required for 125-2H recognition. This region, as examined by IL-18R binding and functional analysis, appeared to be critical for triggering signal transduction through the heterodimeric receptor.