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Pharmacological probes are important tools for exploring disease biology and discovering new therapies. Often molecules of insufficient quality are used instead, leading to spurious and misleading results. The Boehringer Ingelheim open innovation portal opnMe.com addresses this deficiency by sharing extensively validated pharmacological probes with the scientific community.

LFA‐1 (αLβ2) mediates cell‐cell and cell‐extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA‐1 activity is the conformational change of the ligand‐binding αLI domain from low‐affinity (LA), closed form, to the high‐affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA αLI domain have not been described. Here, we report the identification and characterization of a human antibody AL‐57, which binds to the αLI domain in a HA but not LA conformation. AL‐57 was discovered by selection from a human Fab‐displaying library using a locked‐open HA I domain as target. AL‐57 Fab‐phage bound HA I domain‐expressing K562 cells (HA cells) in a Mg2+‐dependent manner. AL‐57 IgG also bound HA cells and PBMCs, activated by Mg2+/EGTA, PMA, or DTT. The binding profile of AL‐57 IgG on PBMCs was the same as that of ICAM‐1, the main ligand of LFA‐1. In contrast, an anti‐αLmurine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL‐57 IgG blocked ICAM‐1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM‐1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA‐induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA‐1‐mediated, adhesive functions. AL‐57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.

Programmed death-1 (PD-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor expressed upon T cell activation. PD-1^–/– animals develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses. Members of the B7 family, PD-L1 and PD-L2, are ligands for PD-1. This study examines the functional consequences of PD-1:PD-L engagementon murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1.Fc-coated beads display dramatically decreased proliferation and IL-2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti-CD28 mAb can overcome PD-1-mediated inhibition by augmenting IL-2 production. However, PD-1:PD-L interactions inhibit IL-2 production even in the presence of costimulation and, thus, after prolonged activation, the PD-1:PD-L inhibitory pathway dominates. Exogenous IL-2 is able to overcome PD-L1-mediated inhibition at all times, indicating that cells maintain IL-2 responsiveness. Experiments using TCR transgenic CD4⁺ or CD8⁺ T cells stimulated with antigen-presenting cells expressing PD-L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8⁺ T cells may be more sensitive to modulation by the PD-1:PD-L pathway because of their intrinsic inability to produce significant levels of IL-2.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor–mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon γ, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand–stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function.

The yeast Saccharomyces cerevisiaecan synthesize, process and secrete higher eukaryotic proteins1–5. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here (1) the synthesis, processing and secretion of light and heavy chains, (2) the glycosylation of heavy chain, (3) the intracellular localization of these foreign proteins by immunofluorescence, and (4) the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia colihas been reported but functional antibodies were not assembled in vivo6,7. Furthermore, only low-level assembly of these chains was found in vitro.

We have developed a strategy to apply genomics and functional genomics-based technologies to the discovery of novel proteins which represent potential therapeutic candidates. The core of this strategy is based on a process for the high-throughput discovery, full-length cDNA cloning and expression of novel human secreted proteins. The availability of full-length cDNA sequences enables sophisticated computational sequence analysis. Expression of the encoded proteins facilitates the direct testing of each gene product in many different bioassays. We expect that a systematic approach to the analysis of this bioassay data, in combination with genetic mapping data and gene expression pattern analysis, will accelerate the discovery and functional analysis of human secreted proteins and, ultimately, the rate of development of therapeutic protein products. Drug Dev. Res. 41:173–179, 1997. © 1997 Wiley-Liss, Inc.

The receptor tyrosine kinaseDtk/Tyro 3/Sky/rse/brt/tifis a member of a new subfamily of receptors that also includesAxl/Ufo/ArkandEyk/Mer.These receptors are characterized by the presence of two immunoglobulin-like loops and two fibronectin type III repeats in their extracellular domains. The structure of the murineDtkgene has been determined. The gene consists of 21 exons that are distributed over 21 kb of genomic DNA. An isoform ofDtkis generated by differential splicing of exons from the 5′ region of the gene. The overall genomic structure ofDtkis virtually identical to that determined for the humanUFOgene. This particular genomic organization is likely to have been duplicated and closely maintained throughout evolution.

We have developed a strategy to apply genomics and functional genomics‐based technologies to the discovery of novel proteins which represent potential therapeutic candidates. The core of this strategy is based on a process for the high‐throughput discovery, full‐length cDNA cloning and expression of novel human secreted proteins. The availability of full‐length cDNA sequences enables sophisticated computational sequence analysis. Expression of the encoded proteins facilitates the direct testing of each gene product in many different bioassays. We expect that a systematic approach to the analysis of this bioassay data, in combination with genetic mapping data and gene expression pattern analysis, will accelerate the discovery and functional analysis of human secreted proteins and, ultimately, the rate of development of therapeutic protein products. Drug Dev. Res. 41:173–179, 1997. © 1997 Wiley‐Liss, Inc.

A gene for murine a heavy chain immunoglobulin has been inserted into a bacterial expression plasmid containing the Eacherichia coli trp promoter and ribosome binding site. A low level expression of μ protein was detected. Secondary structure analysis showed the presence of a hairpin loop burying the μ initiation codon. Alteration of secondary structure at this site by oligonucleotide replacement mutagenesis revealed a correlation between μ expression levels and accessibility of the ribosome binding site. Abolition of secondary structure increased μ protein expression over ninety-fold, to a level approximately equal to that of a trpE-μ fusion protein using the native trpE ribosorae binding site.

Genes for a murine μ heavy chain and a λ light chain immunoglobulin have been inserted into bacterial expression plasmids containing the Escherichia coli trp promoter and ribosoroe binding site. Induction of transcription from the trp promoter results in accumulation of both light and heavy chain polypeptides in appropriate host strains. Both proteins were found as Insoluble products. Following extraction and purification of the immunoglobulin containing fractions, antigen binding activity was recovered. The activity demonstrates essentially the same properties as the antibody from the hybridoma from which the genes were cloned.