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The cell-killing effects of the cytokines TNF-alpha and FasL are mediated by the distinct cell-surface receptors TNFR1, TNFR2 and Fas (also known as CD95/APO-1), which are all members of a receptor superfamily that is important for regulating cell survival. The cytoplasmic regions of TNFR1 and Fas contain a conserved 'death' domain which is an essential component of the signal pathway that triggers apoptosis and activation of the transcription factor NF-kappaB (refs 5,6). Here we report the isolation of a 54K receptor that is a new member of the TNFR superfamily, using the death domain of TNFR1 in a yeast two-hybrid system. This protein, WSL-1, is most similar to TNFR1 itself, particularly in the death-domain region. The gene wsl-1 is capable of inducing apoptosis when transfected into 3T3 and 293 cells, and can also activate NF-kappaB in 293 cells. Like TNFR1, WSL-1 will homodimerize in yeast. WSL-1 also interacts specifically with the TNFR1-associated molecule TRADD. The tissue distribution is very restricted and significantly different from that of Fas and TNFR1.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by ‘third generation chemistries’ may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

DR3 (Ws1, Apo3, LARD, TRAMP, TNFSFR12) is a member of the death domain-containing tumor necrosis factor receptor (TNFR) superfamily, members of which mediate a variety of developmental events including the regulation of cell proliferation, differentiation, and apoptosis. We have investigated the in vivo role(s) of DR3 by generating mice congenitally deficient in the expression of the DR3 gene. We show that negative selection and anti-CD3-induced apoptosis are significantly impaired in DR3-null mice. In contrast, both superantigeninduced negative selection and positive selection are normal. The pre-T-cell receptor-mediated checkpoint, which is dependent on TNFR signaling, is also unaffected in DR3-deficient mice. These data reveal a nonredundant in vivo role for this TNF receptor family member in the removal of self-reactive T cells in the thymus.The tumor necrosis factor receptor (TNFR) superfamily comprise a growing family of type I membrane bound glycoproteins which interact with the TNF family of soluble mediators and type II transmembrane proteins. At least 23 TNFR superfamily members and 17 known ligands have been identified in mammals (reviewed in references 3, 35, and 44). These receptors trigger pleiotropic responses, ranging from apoptosis and differentiation to proliferation, and have been implicated in immune regulation, host defense and lymphoid organ development.Members of the TNFR family are characterized by the presence of varying numbers (three to six) of cysteine-rich repeats in their cytoplasmic domains (52). TNFRs are subdivided based on the presence or absence of a 70-to 80-amino-acid region of homology in the cytoplasmic region called the death domain, through which these receptors trigger apoptosis (20,48). DR3 (also called Ws1, Apo3, TRAMP, LARD, TR3, and TNFRSF12) is one of six death domain-containing TNFR family members (the others are TNFR1, CD95/FAS, DR4, DR5, and DR6) and is the one most closely related to TNFR1. Studies on the TNFR1 crystal structure suggest that ligand binding or receptor overexpression results in receptor trimerization and recruitment of trimeric intracellular signaling molecules (4, 36). DR3, like TNFR1, recruits TNFR1-associated death domain protein (TRADD) and Fas-associated death domain-containing protein (FADD) (5,6,11,12,24) as downstream effectors of apoptosis. These, in turn, interact with caspase 8 (FLICE/MACH) (7, 31), and a cascade of interleukin-1␤-converting enzyme-like cysteine proteases which trigger cell death (13,17,27). DR3 also recruits TRAF2 via TRADD (18,24,29,39) and thus activates the transcription factor, NF-B, that induces the transcription of a number of immune genes (19). In this respect, DR3 (like TNFR1) is capable of inducing both apoptosis and expression of survival/activation genes and is likely to have multiple functions depending on the context of its expression.DR3 was first reported as the only death domain-containing TNFR family member with lymphoid organ-restricted expression (11,24). More recent studies have, however, shown DR3...

SUMMARYNeutralizing monoclonal antibodies raised against type 0 foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type 0 convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed. INTRODUCTIONStudies on the antigenic structure of two picornaviruses, poliovirus and rhinovirus, have been considerably enhanced by the solving of their three-dimensional crystal structure (Hogle et al., 1985;Rossmann et al., 1985) and the isolation and sequence analysis of monoclonal antibody (MAb) escape mutants (Minor et al., 1986;Sherry et al., 1986). Crystallographic studies on footand-mouth disease virus (FMDV), another picornavirus, are also in progress (Fox et al., 1987) and a number of laboratories have generated MAbs capable of neutralizing type O virus. characterized some 30 MAb-resistant mutants of the O1 Kaufbeuren strain of FMDV isolated using five different neutralizing MAbs. Three distinct antigenic sites were involved in virus neutralization. In this context the term antigenic site is used to describe an area of the virus surface which may contain several MAb epitopes; if one epitope is changed and this affects the ability of a second MAb to neutralize the virus then it is considered that the second MAb recognizes an epitope in the same antigenic site as that changed.Recently Stave et al. (1988) have used different MAbs to define three sites of neutralization. In this communication we now use the panel of mutants generated by Xie et al. (1987) to map the binding sites of MAbs generated in different laboratories and then use these antibodies to show the existence of further sites involved in the neutralization of type O FMDV. We have also generated multiple site mutants which are resistant to neutralization at each of two, three or four sites.