Ueli Gubler

A simple and very efficient method for generating cDNA libraries.

A simple method for generating cDNA libraries from submicrogram quantities of mRNA is described. It combines classical first-strand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161-170]. Neither the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used. cDNA thus made can be tailed and cloned without further purification or sizing. Cloning efficiencies can be as high as 10(6) recombinants generated per microgram mRNA, a considerable improvement over earlier methods. Using the fully sequenced 1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.

TWO HUMAN TNF RECEPTORS HAVE SIMILAR EXTRACELLULAR, BUT DISTINCT INTRACELLULAR, DOMAIN SEQUENCES

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

Differential Associations between the Cytoplasmic Regions of the Interleukin-12 Receptor Subunits β1 and β2 and JAK Kinases

The role of the cytoplasmic regions of interleukin-12 receptors (IL-12R) β1 and β2 in stimulating proliferation was examined. The transmembrane and cytoplasmic regions of IL-12Rβ1 or IL-12Rβ2 were fused to the extracellular domain of the epidermal growth factor (EGF) receptor, yielding chimeric receptors E12R1 and E12R2, respectively. These chimeras were stably transfected into BaF3 cells, a factor-dependent murine pro-B cell line. Only E12R2 or E12R1+E12R2 transfectants were capable of EGF-dependent proliferation. EGF-dependent phosphorylation of E12R2, JAK2, Tyk2, and STAT3 was observed. JAK2 was phosphorylated in E12R1-, E12R2-, and E12R1+E12R2-expressing cells. However, direct associations were detectable only between E12R2 and JAK2. Tyk2 phosphorylation was observed only in cells expressing E12R1 or E12R1+E12R2. In parallel with this activation pattern, direct interactions only between Tyk2 and E12R1 were demonstrable. Phosphorylation of STAT3 was observed in cells expressing E12R1, E12R2, and E12R1+E12R2. The expression levels of STAT4 protein in BaF3 cells are undetectable by the methods employed here; therefore, STAT4 phosphorylation was not observed. Taken together, the data indicate that differential interactions take place between the cytoplasmic regions of the two IL-12R subunits and JAK2/Tyk2 and that the cytoplasmic region of IL-12Rβ2 alone is capable of delivering a proliferative signal.

Mycobacterium tuberculosis infection induces il12rb1 splicing to generate a novel IL-12Rβ1 isoform that enhances DC migration

RNA splicing is an increasingly recognized regulator of immunity. Here, we demonstrate that after Mycobacterium tuberculosis infection (mRNA) il12rb1 is spliced by dendritic cells (DCs) to form an alternative (mRNA) il12rb1Δtm that encodes the protein IL-12Rβ1ΔTM. Compared with IL-12Rβ1, IL-12Rβ1ΔTM contains an altered C-terminal sequence and lacks a transmembrane domain. Expression of IL-12Rβ1ΔTM occurs in CD11c+ cells in the lungs during M. tuberculosis infection. Selective reconstitution of il12rb1−/− DCs with (mRNA) il12rb1 and/or (mRNA) il12rb1Δtm demonstrates that IL-12Rβ1ΔTM augments IL-12Rβ1-dependent DC migration and activation of M. tuberculosis-specific T cells. It cannot mediate these activities independently of IL12Rβ1. We hypothesize that M. tuberculosis-exposed DCs express IL-12Rβ1ΔTM to enhance IL-12Rβ1-dependent migration and promote M. tuberculosis–specific T cell activation. IL-12Rβ1ΔTM thus represents a novel positive-regulator of IL12Rβ1-dependent DC function and of the immune response to M. tuberculosis.

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.

Evidence for Multiple Sites of Interaction between IL‐12 and Its Receptor

We have previously described the identification of a protein, now designated IL-12R beta 1, that binds 125I-huIL-12 with a Kd of about 10 nM, corresponding to the low affinity 125I-huIL-12 binding sites seen on PHA-activated human lymphoblasts. Using expression cloning techniques, we have recently identified an additional IL-12-binding protein subunit, IL-12R beta 2, which binds 125I-huIL-12 with a Kd of about 5 nM when expressed alone in COS-7 cells. Coexpression of IL-12R beta 1 and IL-12R beta 2 in COS-7 cells results in formation of two classes of 125 I-huIL-12-binding sites with Kds of about 50 pM and 5 nM. Mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to human IL-12R beta 1, but did not inhibit binding to human IL-12R beta 2. In contrast, anti-huIL-12 monoclonal antibody 20C2, which does not block 125I-huIL-12 binding to human IL-12R beta 1, completely blocked binding to human IL-12R beta 2. These results demonstrate that two classes of IL-12 inhibitors, one that primarily blocks IL-12/IL-12R beta 1 interaction (e.g., mo(p40)2), and one that primarily blocks IL-12/IL-12R beta 2 interaction (e.g., 20C2), can be identified.

Molecular Biology of lnterleukin‐12 Receptors

IL-12 receptors are expressed primarily on activated T and NK cells. Using labeled IL-12, three classes of IL-12-binding sites have been identified on human PHA-activated lymphoblasts. Their Kd values are 5-20 pM (high affinity), 50-200 pM (intermediate affinity), and 2-6 nM (low affinity). Using a monoclonal antibody, a cDNA encoding a human IL-12 receptor was isolated by expression cloning. The homologous mouse cDNA was isolated by cross hybridization. These proteins are gp130-like members of the cytokine receptor superfamily. Individually, however they bind IL-12 with low affinity. An expression cloning approach was undertaken to isolate an additional human IL-12 receptor component that would act as a high-affinity converter. A cDNA encoding another IL-12 receptor was isolated. The mouse homologue was obtained by cross hybridization. These IL-12 receptors are also gp130-like cytokine receptor superfamily members. Because these two types of IL-12 receptors have the general makeup of beta-type cytokine receptor subunits, they are designated as IL-12R beta 1 and IL-12R beta 2. Coexpression of IL-12R beta 1 and IL-12R beta 2 leads to the formation of high-affinity IL-12-binding sites and reconstitution of IL-12-dependent signaling.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Allosteric antibody inhibition of human hepsin protease

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation.

Abstract 4827: Humanized hepsin neutralizing antibody RO5486055 inhibits tumor growth and leads to accumulation of hepsin substrate laminin-332

Hepsin is overexpressed in prostate and other cancers where it is implicated in promoting tumor growth, invasion, and metastasis. To further our understanding of the role of hepsin in prostate and breast cancer, a fully humanized monoclonal antibody that recognizes human and cynomolgus hepsin has been developed. RO5486055 selectively binds to hepsin but not related type II transmembrane serine proteases, and neutralizes hepsin serine protease activity with an IC 50 in the single digit nM range. In LNCaP prostate cancer cells, both shRNA knockdown of the hepsin gene and RO5486055 treatment cause a similar accumulation of the β3 chain of laminin-332, a known hepsin substrate. With ip administration in mice, RO5486055 demonstrates dose-dependent exposure and a long serum half-life of 168-406 hours. RO5486055 attenuates tumor growth in the LNCaP prostate cancer and T-47D breast cancer xenograft models, but not in the CRW-22Rv1 prostate or MCF-7 breast cancer xenografts. In these models, it has been shown that the level of the β3 chain of laminin-332 detected by Western blot analysis predicts sensitivity to RO5486055-mediated growth inhibition. Moreover, accumulation of the β3 chain during RO5486055 treatment correlates with antitumor activity. In 8/10 hepsin-expressing patient-derived prostate tumors, an inverse correlation between hepsin and β3 chain expression is observed. The β3 chain of laminin-332 may therefore be useful as both a predictive and response biomarker for anti-hepsin therapy. RO5486055-mediated tumor growth inhibition is enhanced by combination with the EGFR-targeted antibody cetuximab in LNCaP prostate cancer xenografts, and by combination with hormone withdrawal in T-47D breast cancer xenografts. These preclinical results suggest that hepsin-directed therapy could be effective in prostate and breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4827. doi:1538-7445.AM2012-4827