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Upon exposing mammalian tissues to hypoxia, expression of a number of physiologically important genes such as erythropoietin and vascular endothelial growth factor (VEGF) increases. The key regulator for this oxygen-dependent gene expression is the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of an alpha and a beta subunit. Both HIF-1 subunits are widely expressed in the cells and tissue of vertebrates, flies, fishes, worms and probably most other species. The beta subunit (also termed ARNT, aryl hydrocarbon receptor nuclear translocator) is abundantly expressed in an oxygen-independent manner. On the other hand, HIF-1alpha cannot be detected above a critical partial pressure of oxygen when it is subjected to rapid ubiquitinylation and proteasomal degradation. Hypoxic exposure leads to stabilization of HIF-1alpha protein and subsequent activation of HIF-1-dependent target genes. HIF-1 is not only a master regulator of oxygen homeostasis, it also appears to play a key role in tumor development as well as cardiovascular and ischemic diseases. Genetic modulation of HIF-1alpha activity in vivo may therefore represent a novel therapeutic approach to these disorders. In this overview, we report on the generation of HIF-1alpha overexpressing HeLa cell lines and demonstrate the feasibility of normoxic HIF-1 gene transfer in vitro and in vivo thereby identifying the limiting steps for full activation of HIF-1.

Hypoxia-inducible factor (HIF)-1alpha is the oxygen-sensitive subunit of HIF-1, a transcriptional master regulator of oxygen homeostasis. Oxygen-dependent prolyl hydroxylation targets HIF-1alpha for ubiquitinylation and proteasomal degradation. Unexpectedly, we found that exposing mice to elevated temperatures resulted in a strong HIF-1alpha induction in kidney, liver, and spleen. To elucidate the molecular mechanisms responsible for this effect, HepG2 hepatoma cells were exposed to different temperatures (34-42 degrees C) under normoxic (20% O(2)) or hypoxic (3% O(2)) conditions. Heat was sufficient to stabilize mainly a phosphatase-resistant, low molecular weight form of HIF-1alpha (termed HIF-1alpha(a)). Heat-induced HIF-1alpha(a) accumulated in the nucleus but neither bound to DNA nor trans-activated reporter or target gene expression, demonstrating the need for post-translational modifications for these functions. The protein banding pattern of heat-induced HIF-1alpha in immunoblot analyses was clearly distinct from the HIF-1alpha pattern after prolyl hydroxylase inhibition (by hypoxia or iron chelation/replacement) or following proteasome inhibition, suggesting that heat stabilizes HIF-1alpha by a novel mechanism. Inhibition of the ATP-dependent chaperone activity of HSP90 by novobiocin or geldanamycin prevented heat-induced as well as hypoxia-induced HIF-1alpha accumulation, indicating a common role of the HSP90 chaperone activity in HIF-1alpha stabilization by these two environmental parameters.

The PAS domain is a versatile protein fold found in many archaeal, bacterial, and plant proteins capable of sensing environmental changes in light intensity, oxygen concentration, and redox potentials. The oxygen sensor FixL from Rhizobium species contains a heme-bearing PAS domain and a histidine kinase domain that couples sensing to signaling. We identified a novel mammalian PAS protein (PASKIN) containing a domain architecture resembling FixL. PASKIN is encoded by an evolutionarily conserved single-copy gene which is ubiquitously expressed. The human PASKIN and mouse Paskin genes show a conserved intron-exon structure and share their promoter regions with another ubiquitously expressed gene that encodes a regulator of protein phosphatase-1. The 144-kDa PASKIN protein contains a PAS region homologous to the FixL PAS domain and a serine/threonine kinase domain which might be involved in signaling. Thus, PASKIN is likely to function as a mammalian PAS sensor protein.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.

The erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

Antibody conjugates have broad utility in basic, preclinical, and clinical applications. Conventional antibody conjugation through the amine group of lysine or the thiol group of cysteine residues yields heterogeneous products of undefined stoichiometry and considerable batch-to-batch variability. To preserve the two hallmarks of the antibody molecule, precision and predictability, methods that enable site-specific antibody conjugation are in high demand. On the basis of a mammalian cell expression system, we describe the utilization of the 21st natural amino acid selenocysteine for the generation of IgG and Fab molecules with unique nucleophilic reactivity that affords site-specific conjugation to electrophilic derivatives of biotin, fluorescein, and poly(ethylene glycol). The resulting antibody conjugates were found to fully retain their antigen binding capability and, in the case of IgG, the ability to mediate effector functions. Gain of function was demonstrated in vitro and in vivo. While these antibody conjugates are relevant for a variety of proteomic, diagnostic, and therapeutic applications, they also constitute a proof of principle for the generation of molecularly defined antibody-drug conjugates and radioimmunoconjugates. Compared to other site-specific antibody conjugation methods, selenocysteine interface technology (i) only involves a minor modification at the C-terminus that does not interfere with disulfide bridges, (ii) does not require activation, and (iii) generates unique 1:1 stoichiometries of biological and chemical components. Collectively, our method affords the generation of highly defined antibody conjugates with broad utility from proteomic applications to therapeutic intervention.

Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin α 4 β 1 . Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin α 4 β 1 and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.

A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin alpha(4)beta(1)-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin alpha(4)beta(1). The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.

Aryl hydrocarbon receptor (AhR) and hypoxiainducible factor-1α (HIF-1α) are conditionally regulated transcription factor subunits that form heterodimeric complexes with their common partner, AhR nuclear translocator (ARNT/HIF-1β). Whereas the environmentally toxic compound 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) initiates the trans-activation activity of AhR:ARNT/HIF-1β, hypoxic exposure stabilizes HIF-1α and functionally activates the HIF-1α:ARNT/HIF-1β complex. To analyze a possible crosstalk between these two pathwaysin vivo, rats were given dioxin orally and/or were exposed to carbon monoxide (CO), causing functional anemia. We found that exposure to CO inhibited the xenobiotic response while dioxin application had no significant negative impact on hypoxia-mediated gene transcription.

The key elements of circadian clockwork and oxygen homeostasis are the PAS protein family members PER and CLOCK and hypoxia-inducible factor 1alpha (HIF-1alpha). The PAS domain serves as an interface for protein-protein interactions. We asked whether a cross-talk exists between the PAS components of hypoxic and circadian pathways. We found several isoforms of PER1 protein that exhibit tissue-specific size differences. In the mouse brain, a predominantly nuclear 48 kDa isoform that followed a daily rhythm was observed. The 48 kDa form was found in the nuclear fractions derived from mouse liver, Swiss3T3 fibroblasts, and N2A neuroblastoma cells. In mouse kidney and human 293 kidney cells, a 55 kDa PER1 form was detected. CLOCK was observed as a predicted 100 kDa protein in rat-1 cells and in all analyzed mouse tissues including brain, liver, kidney, and spleen. In contrast to PER1, CLOCK protein expression was not rhythmic. Exposure to hypoxia led to increased PER1 and CLOCK protein levels in mice. Based on coimmunoprecipitation experiments that showed protein-protein interaction between PER1 and the alpha subunit of HIF-1, we suggest that these hypoxic effects may be modulated by HIF-1alpha.-Chilov, D., Hofer, T., Bauer, C., Wenger, R. H., Gassmann, M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.

The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the entire ccmH gene was deleted failed to synthesize both indigenous and foreign c-type cytochromes. However, deletion of the C-terminal hydrophilic domain homologous to CycH of other gram-negative bacteria affected neither the biogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium japonicum cytochrome c 550. This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analysis showed that this domain is periplasmic. Site-directed mutagenesis of the cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cysteine is required for cytochrome c maturation during anaerobic growth. The deficiency of the point mutants was complemented when 2-mercapto-ethanesulfonic acid was added to growing cells; other thiol compounds did not stimulate cytochrome c formation in these strains. We propose a model for the reaction sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation.

Introduction IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the known IL-2-based immunocytokines has yet progressed to pivotal clinical trials due to various constraints in their design; in particular, the fusion of two wild-type IL-2 moieties to the antibody and retained FcgR binding of IgG-based immunocytokines. This design results in 1) high affinity binding with pM affinity to IL-2Raβγ on immune cells compromising tumor targeting and inducing rapid systemic clearance and short half-life; 2) high affinity for CD25 (IL-2Ra) expressed on pulmonary vascular endothelium contributing to pulmonary toxicity; and 3) preferential activation of Tregs over immune effectors. Here we describe a novel class of monomeric tumor-targeted immunocytokines where a single, engineered IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For tumor targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA-IL2v recognizes a membrane proximal epitope of human carcinoembryonic antigen (CEA) and binds preferentially to membrane-bound CEA, but not shed CEA. Methods CEA- and FAP-IL2v were produced as recombinant proteins and their activity tested oneffector cells by assessing the activation of P-STAT5, cell proliferation, sensitivity to Fas-induced apoptosis, expression of activation markers and cytokine release upon treatment. Safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were analyzed in SCID and fully immunocompetent C57Bl/6 mice as single agent and in combination with trastuzumab and cetuximab. Tumor targeting was investigated in the orthotopic syngeneic Renca renal cell cancer tumor model in Balb/c mice by SPECT imaging. Results FAP- and CEA-IL2v completely lack binding to CD25, but retain IL-Rβγ binding, and show pM binding affinity to respective antigens, FAP on fibroblasts and CEA on tumor cells. As consequence of abolished binding to CD25 these molecules do not preferentially activate Tregs. The treatment of effector cells with IL2v reduces their sensitivity for Fas-mediated apoptosis (also known as activation induced cell death) as compared to wild-type IL-2 based immunocytokine. IL-2Rβγ bioactivity was retained and FAP- and CEA-IL2v activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers, cell proliferation and cytokine release. Furthermore, CEA-IL2v and FAP-IL2v enhanced the cytotoxic activity of NK cells when combined with ADCC-competent antibodies. Mechanism of action studies in fully immunocompetent mice showed that the molecules strongly expand and activate NK, CD8+ T cells and gd T cells (up to 100-fold) and skew the CD4:CD8 ratio strongly towards CD8+ T cells in the peripheral blood, lymphoid tissues, and in the tumor. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite a higher exposure and circulatory half-life than the analogous IL-2 based immunocytokine. MicroSPECT/CT imaging with radioactively labeled FAP-IL2v revealed good FAP-mediated tumor targeting in the orthotopic syngeneic Renca model with low normal tissue uptake and low accumulation in lymphoid tissues, contrary to analogous IL-2 based immunocytokine that showed preferential targeting to lymphoid tissue. Studies in tumor-bearing mice showed dose-dependent anti-tumor efficacy of FAP-IL2v and CEA-IL2v in syngeneic models. Additional studies in xenograft models in SCID mice transgenic for human CD16A showed that CEA-IL2v strongly enhances the antitumor efficacy and/or survival mediated by ADCC-competentantibodies, including trastuzumab and cetuximab. Conclusion CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 binding, monovalency and high-affinity tumor-targeting as compared to classical IL-2-based immunocytokines. They retain capacity to activate and expand NK and CD8+ effector T cells through IL-2Rβγ in the periphery and the tumor microenvironment. These data support their further nonclinical and clinical investigation for immunotherapy of cancer. Clinical trials with CEA-IL2v are foreseen in 2014. Disclosures: Klein: Roche Glycart AG: Employment. Waldhauer:Roche: Employment. Nicolini:Roche: Employment. Dunn:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Danny:Roche: Research Funding. Boerman:Roche: Research Funding. Nayak:Roche: Employment. Herter:Roche: Employment. Van Puijenbroek:Roche: Employment. Ast:Roche: Employment. Hofer:Roche: Employment. Hosse:Roche: Employment. Lang:Roche: Employment. Neumann:Roche: Employment. Kettenberger:Roche: Employment. Neubauer:Roche: Employment. Gorr:Roche: Employment. Tuerck:Roche: Employment. Evers:Roche: Employment. Gerdes:Roche: Employment. Levitsky:Roche: Employment. Bacac:Roche: Employment. Moessner:Roche: Employment. Umana:Roche: Employment, Equity Ownership.