Single Polypeptide Chain Research Articles

Identification, Expression and Preliminary Characterization of a Recombinant Bifunctional Enzyme of Photobacterium damselae subsp. piscicida with Glutamate Decarboxylase/Transaminase Activity

Glutamate decarboxylase catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), contributing to pH homeostasis through proton consumption. The reaction is the first step toward the GABA shunt. To date, the enzymes involved in the glutamate metabolism of Photobacterium damselae subsp. piscicida have not been elucidated. In this study, an open reading frame of P. damselae subsp. piscicida, showing homology to the glutamate decarboxylase or putative pyridoxal-dependent aspartate 1-decarboxylase genes, was isolated and cloned into an expression vector to produce the recombinant enzyme. Preliminary gas chromatography-mass spectrometry characterization of the purified recombinant enzyme revealed that it catalyzed not only the decarboxylation of glutamate but also the transamination of GABA. This enzyme of P. damselae subsp. piscicida could be bifunctional, combining decarboxylase and transaminase activities in a single polypeptide chain.

Proteins with calmodulin-like domains: structures and functional roles.

The appearance of modular proteins is a widespread phenomenon during the evolution of proteins. The combinatorial arrangement of different functional and/or structural domains within a single polypeptide chain yields a wide variety of activities and regulatory properties to the modular proteins. In this review, we will discuss proteins, that in addition to their catalytic, transport, structure, localization or adaptor functions, also have segments resembling the helix-loop-helix EF-hand motifs found in Ca2+-binding proteins, such as calmodulin (CaM). These segments are denoted CaM-like domains (CaM-LDs) and play a regulatory role, making these CaM-like proteins sensitive to Ca2+ transients within the cell, and hence are able to transduce the Ca2+ signal leading to specific cellular responses. Importantly, this arrangement allows to this group of proteins direct regulation independent of other Ca2+-sensitive sensor/transducer proteins, such as CaM. In addition, this review also covers CaM-binding proteins, in which their CaM-binding site (CBS), in the absence of CaM, is proposed to interact with other segments of the same protein denoted CaM-like binding site (CLBS). CLBS are important regulatory motifs, acting either by keeping these CaM-binding proteins inactive in the absence of CaM, enhancing the stability of protein complexes and/or facilitating their dimerization via CBS/CLBS interaction. The existence of proteins containing CaM-LDs or CLBSs substantially adds to the enormous versatility and complexity of Ca2+/CaM signaling.

Identification of a novel lactose oxidase in Myrmecridium flexuosum NUK-21.

Lactobionic acid (O‐β‐galactosyl‐(1‐4)‐gluconic acid) (LBA) is a high‐value lactose derivative, produced via oxidation of the reducing terminal of lactose. LBA can be produced by fermentation using certain microorganisms, although subsequent purification is challenging. Therefore, we have attempted to identify an enzyme for possible use in LBA production. Here, we purified a novel lactose oxidase (LOD) to homogeneity from a wheat bran culture of a soil‐isolated fungal strain, Myrmecridium flexuosum NUK‐21. Maximal activity was observed on the wheat bran solid culture after 3 days of NUK‐21 growth, following release from cells at 0.66 unit·mL −1 culture filtrate. This new sugar oxidase was composed of a single polypeptide chain with a molecular mass of 47.2 kDa and was found to contain 2.0 zinc ions per mole of enzyme but no flavin adenine dinucleotide or heme. This enzyme was stable in the pH range 5.5–9.0, with an optimal reaction pH of 7.5. Its optimal reaction temperature was 40 °C, and it was stable up to 50 °C for 1 h at pH 7.5. LOD oxidized disaccharides with reducing‐end glucosyl residues linked by an α or β‐1,4 glucosidic bond. The relative activity of LOD toward lactose, cellobiose and maltose was 100 : 83 : 4, respectively. To the best of our knowledge, this is the first report on the discovery of an LOD based on coenzyme moiety and enzyme substrate specificity.

Intradermal Application of Crotamine Induces Inflammatory and Immunological Changes In Vivo.

Crotamine is a single-chain polypeptide with cell-penetrating properties, which is considered a promising molecule for clinical use. Nevertheless, its biosafety data are still scarce. Herein, we assessed the in vivo proinflammatory properties of crotamine, including its local effect and systemic serum parameters. Sixty male Wistar rats were intradermically injected with 200, 400 and 800 µg crotamine and analyzed after 1, 3 and 7 days. Local effect of crotamine was assessed by determination of MPO and NAG activities, NO levels and angiogenesis. Systemic inflammatory response was assessed by determination of IL-10, TNF-α, CRP, NO, TBARS and SH groups. Crotamine induced macrophages and neutrophils chemotaxis as evidenced by the upregulation of both NAG (0.5–0.6 OD/mg) and MPO (0.1–0.2 OD/mg) activities, on the first and third day of analysis, respectively. High levels of NO were observed for all concentrations and time-points. Moreover, 800 μg crotamine resulted in serum NO (64.7 μM) and local tissue NO (58.5 μM) levels higher or equivalent to those recorded for their respective histamine controls (55.7 μM and 59.0 μM). Crotamine also induced a significant angiogenic response compared to histamine. Systemically, crotamine induced a progressive increase in serum CRP levels up to the third day of analysis (22.4–45.8 mg/mL), which was significantly greater than control values. Crotamine (400 μg) also caused an increase in serum TNF-α, in the first day of analysis (1095.4 pg/mL), however a significant increase in IL-10 (122.2 pg/mL) was also recorded for the same time-point, suggesting the induction of an anti-inflammatory effect. Finally, crotamine changed the systemic redox state by inducing gradual increase in serum levels of TBARS (1.0–1.8 μM/mL) and decrease in SH levels (124.7–19.5 μM/mL) throughout the experimental period of analysis. In summary, rats intradermally injected with crotamine presented local and systemic acute inflammatory responses similarly to histamine, which limits crotamine therapeutic use on its original form.

Evaluation of the oral immunogenicity of M cell-targeted tetravalent EDIII antigen for development of plant-based edible vaccine against dengue infection

Four serotypes of the dengue virus that can cause severe disease in humans greatly increases the complexity of vaccine development. In this study, we evaluated an ‘all-in-one’ tetravalent dengue vaccine candidate as an alternative to the classical four viruses ‘cocktail’ vaccination approach. A single polypeptide chain of the tetra-EDIII comprising all four serotypes fused to the M-cell target ligand peptide (tEDIII-Co1) was constructed and expressed using a rice expression system under the control of a strong inducible promoter (rice amylase 3D: Ramy3D) and rice 3′ untranslated region (UTR). The tEDIII-Co1 was constructed using wild type or plant codon optimized gene sequence to compare the expression levels. The codon optimized gene combined with ER-localization showed higher and more stable expression than codon-optimization alone or the wild type. To test the oral immunogenicity of this construct, the transgenic rice cells containing 100 µg of tEDIII-Co1 were fed to mice and the antigen specific IgG and IgA antibodies analysed in sera or fecal extracts. The tEDIII fused to Co1 induced strong antibody responses in the absence of any adjuvants. We observed the strongest antigen-specific B and T cell responses in animals immunized with tEDIII-Co1. Taken together, this simple tetravalent EDIII-based fusion protein with the M cell ligand could has potential as an alternative dengue vaccine strategy against all four serotypes.

PlantMP: a database for moonlighting plant proteins.

Moonlighting proteins are single polypeptide chains capable of executing two or more distinct biochemical and/or biological functions. Here, we describe the development of PlantMP, which is a manually curated online-based database of plant proteins that are known to `moonlight’. The database contains searchable UniProt IDs and names, canonical and moonlighting functions, gene ontology numbers, plant species as well as links to the PubMed indexed articles. Proteins homologous to experimentally confirmed moonlighting proteins from the model plant Arabidopsis thaliana are provided as a separate list of `likely moonlighters’. Additionally, we also provide a list of predicted Arabidopsis moonlighting proteins reported in the literature. Currently, PlantMP contains 110 plant moonlighting proteins, 10 `likely moonlighters’ and 27 `predicted moonlighters’. Organizing plant moonlighting proteins in one platform enables researchers to conveniently harvest plant-specific raw and processed data such as the molecular functions, biological roles and structural features essential for hypothesis formulation in basic research and for biotechnological innovations.

The power of the force: mechano-physiology of the giant titin.

Titin - the largest protein in the human body - spans half of the muscle sarcomere from the Z-disk to the M-band through a single polypeptide chain. More than 30 000 amino acid residues coded from a single gene (TTN, in humans Q8WZ42) form a long filamentous protein organized in individual globular domains concatenated in tandem. Owing to its location and close interaction with the other muscle filaments, titin is considered the third filament of muscle, after the thick-myosin and the thin-actin filaments.

Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels

The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective β subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshβ or mdLhβ mature protein coding sequences to produce a “tethered” polypeptide with the β-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshβ and mdLhβ was determined by immunofluorescence (IF) for Fshβ and Lhβ on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshβ or mdLhβ, and the tethered proteins mdFshβα or mdLhβα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.

Potential Use of Genetically Engineered Variable Lymphocyte Receptor B Specific to Avian Influenza Virus H9N2.

The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2-specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine-serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.

The Extreme Structural Plasticity in the CYP153 Subfamily of P450s Directs Development of Designer Hydroxylases.

CYP153s are bacterial class I P450 enzymes traditionally described as alkane hydroxylases with a high terminal regioselectivity. They have been more recently shown to also catalyze hydroxylations at nonactivated carbon atoms of small heterocycles. The aim of our work was to perform an extensive characterization of this subfamily in order to deliver a toolbox of CYP153 enzymes for further development as biocatalysts. Through the screening of recently sequenced bacterial genomes, 20 CYP153s were selected, comprising 17 single monooxygenase domains and three multidomain variants, where the monooxygenase domain is naturally fused to its redox partners in a single polypeptide chain. The 20 novel variants were heterologously expressed, and their activity was screened toward octane and small heterocycles. A more extended substrate characterization was then performed on three representative candidates, and their crystal structures were unveiled and compared with those of the known CYP153A7 and CYP153A33. The tested enzymes displayed a wide range of activities, ranging from Ω and Ω-1 hydroxylations of lauric acid to indigo-generating indole modification. The comparative analysis highlighted a conserved architecture and amino acid composition of the catalytic core close to the heme, while showing a huge degree of structural plasticity and flexibility in those regions hosting the substrate recognition sites. Although dealing with this type of conformational variability adds a layer of complexity and difficulty to structure-based protein engineering, such diversity in substrate acceptance and recognition promotes the investigated CYP153s as a prime choice for tailoring designer hydroxylases.

OpuF, a New Bacillus Compatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain.

The accumulation of compatible solutes is a common defense of bacteria against the detrimental effects of high osmolarity. Uptake systems for these compounds are cornerstones in cellular osmostress responses because they allow the energy-preserving scavenging of osmostress protectants from environmental sources. Bacillus subtilis is well studied with respect to the import of compatible solutes and its five transport systems (OpuA, OpuB, OpuC, OpuD, and OpuE), for these stress protectants have previously been comprehensively studied. Building on this knowledge and taking advantage of the unabated appearance of new genome sequences of members of the genus Bacillus, we report here the discovery, physiological characterization, and phylogenomics of a new member of the Opu family of transporters, OpuF (OpuFA-OpuFB). OpuF is not present in B. subtilis but it is widely distributed in members of the large genus Bacillus OpuF is a representative of a subgroup of ATP-binding cassette (ABC) transporters in which the substrate-binding protein (SBP) is fused to the transmembrane domain (TMD). We studied the salient features of the OpuF transporters from Bacillus infantis and Bacillus panaciterrae by functional reconstitution in a B. subtilis chassis strain lacking known Opu transporters. A common property of the examined OpuF systems is their substrate profile; OpuF mediates the import of glycine betaine, proline betaine, homobetaine, and the marine osmolyte dimethylsulfoniopropionate (DMSP). An in silico model of the SBP domain of the TMD-SBP hybrid protein OpuFB was established. It revealed the presence of an aromatic cage, a structural feature commonly present in ligand-binding sites of compatible solute importers.IMPORTANCE The high-affinity import of compatible solutes from environmental sources is an important aspect of the cellular defense of many bacteria and archaea against the harmful effects of high external osmolarity. The accumulation of these osmostress protectants counteracts high-osmolarity-instigated water efflux, a drop in turgor to nonphysiological values, and an undue increase in molecular crowding of the cytoplasm; they thereby foster microbial growth under osmotically unfavorable conditions. Importers for compatible solutes allow the energy-preserving scavenging of osmoprotective and physiologically compliant organic solutes from environmental sources. We report here the discovery, exemplary physiological characterization, and phylogenomics of a new compatible solute importer, OpuF, widely found in members of the Bacillus genus. The OpuF system is a representative of a growing subgroup of ABC transporters in which the substrate-scavenging function of the substrate-binding protein (SBP) and the membrane-embedded substrate translocating subunit (TMD) are fused into a single polypeptide chain.

The effect of Jun dimerization on neurite outgrowth and motif binding

Axon regeneration is a necessary step toward functional recovery after spinal cord injury. The AP-1 transcription factor c-Jun has long been known to play an important role in directing the transcriptional response of Dorsal Root Ganglion (DRG) neurons to peripheral axotomy that results in successful axon regeneration. Here we performed ChIPseq for Jun in mouse DRG neurons after a sciatic nerve crush or sham surgery in order to measure the changes in Jun's DNA binding in response to peripheral axotomy. We found that the majority of Jun's injury-responsive changes in DNA binding occur at putative enhancer elements, rather than proximal to transcription start sites. We also used a series of single polypeptide chain tandem transcription factors to test the effects of different Jun-containing dimers on neurite outgrowth in DRG, cortical and hippocampal neurons. These experiments demonstrated that dimers composed of Jun and Atf3 promoted neurite outgrowth in rat CNS neurons as well as mouse DRG neurons. Our work provides new insight into the mechanisms underlying Jun's role in axon regeneration.

Stability of total prostate-specific antigen and free prostate-specific antigen after 10 years' storage.

PSA is a serine protease composed of 240 amino acids in a single polypeptide chain and is a routine parameter in prostate cancer diagnostics. The aim of our study was to test the long-term stability of tPSA and fPSA after 10 years' storage at -80°C. We analyzed two aliquots from 55 serum samples. The first was assayed in routine testing at the time of establishing the diagnosis. The second was thawed for further testing after approximately 10 years' storage at -80°C. The mean of storage time was 10.41 years (min-max: 9.35-11.40 years). We compared the results of tPSA and fPSA. We calculated the fPSA/tPSA ratio and compared the results of clinical evaluation. Serum tPSA and fPSA levels were assayed using chemiluminescent kits Access Hybritech PSA and free PSA. All measurements were performed using the instrument UniCel® DxI 800. tPSA decreased 3.59% on average with a correlation r=0.9213, and fPSA increased at an average of 2.41% with a correlation r=0.9338. The fPSA/tPSA ratio increased 0.80% on average with a correlation r=0.9174. On clinical evaluation, five samples had fallen to a less malignant category and three samples had risen to a higher malignant category compared with the original results. The stability of tPSA and fPSA levels in serum is sufficient after 10 years' storage at -80°C. Calculation of the fPSA/tPSA ratio is not recommended due to the change in the category of malignancy of 15% of the samples.

Crystal structure of a novel Kunitz type inhibitor, alocasin with anti-Aedes aegypti activity targeting midgut proteases.

The pesticidal properties of many Kunitz-type inhibitors have been reported previously; however, the mechanism of action is not well established. In this study, the activity of alocasin against Aedes aegypti is demonstrated and the structure-activity relationship of this Kunitz-type inhibitor is explained through X-ray structure analyses. Alocasin was purified from mature rhizomes of Alocasia as a single polypeptide chain of ∼ 20 kDa. The structure at 2.5 Å resolution revealed a Kunitz-type fold, but variation in the loop regions makes this structure unique; one loop with a single disulfide bridge is replaced by a long loop with two bridges. Alignment of homologous sequences revealed that this long loop contains a conserved Arg residue and modeling studies showed interaction with the catalytic Ser residue of trypsin-like enzymes. The anti-Aedes aegypti activity of alocasin is examined and discussed in detail. The in vitro activity of alocasin against midgut proteases of Aedes aegypti showed profound inhibition. Further, morphological changes in larvae upon treatment with alocasin revealed its activity against Ae. aegypti. Docking studies of alocasin with trypsin (5G1), a midgut protease involved in the development cycle and blood meal digestion, illustrated its insecticidal activity. The three-dimensional structure of alocasin was determined and its structure-function relationship established for its anti Ae. aegypti activity. © 2018 Society of Chemical Industry.

Abstract 2716: Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies in vivo

Abstract Members of the tumor necrosis factor (TNF) superfamily of costimulatory receptors have emerged as promising targets for modulation of tumor infiltrating lymphocytes. Among these, 4-1BB (CD137) has a prominent role in promoting survival and expansion of cytotoxic CD8 T cells as well as activation of CD4 T cells, NK cells, and antigen presenting cells. The clinical relevance of 4-1BB agonism is currently being evaluated for two antibodies: urelumab (IgG4) and utomilumab (IgG2). However, it has been established that for the trimeric TNF receptor family, the homotrimeric ligands are superior activators compared to bivalent agonistic antibodies. Here we describe a prototype 4-1BB agonist consisting of three human 4-1BB ligands linked together as a single polypeptide chain and fused to the C-terminus of human IgG1 Fc (STIM-41BBL). Compared to a clinically relevant human 4-1BB antibody, STIM-41BBL shows increased activation of 4-1BB in a NF-kappa B reporter cell line. STIM-4-1BBL also shows augmented cytokine release compared to the antibody in CD8 T cells. Furthermore, we have developed a mouse STIM-41BBL and like the human counterpart, the ligand fusion induced greater cytokine release than an agonistic mouse 4-1BB antibody (IgG2a) in CD8 T cells. We have also introduced mutations (D265A/N297A) to the mouse 4-1BB antibody to make it effectorless in order to better approximate the clinical 4-1BB antibodies. Subsequently, we demonstrate that the murine STIM-41BBL had greater anti-tumor activity than the 4-1BB antibody in a MC38 tumor model. These results support the continued development of a 4-1BB ligand based therapeutic and suggests the greater potential of a STIM-41BBL fusion to a targeting antibody for more precise activation of immune system. Citation Format: Eric Tam, Galina Craig, Sara Ghassemifar, Alexander Koshkaryev, Sara Movassaghian, Cormac Cosgrove, James Sampson, Daryl Drummond, Andreas Raue. Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2716.

Abstract 5553: A novel immunocytokine for the treatment of cancer

Abstract Interleukin-12 (IL12) is an immunomodulatory cytokine, which offers unique opportunities for cancer therapy due to its stimulatory function on cell-mediated immunity and its anti-angiogenic activity. IL12 was shown to polarize CD4+ T cells into a TH1 type and to be a key activator of NK cells and CD8+ T cells. It also induces the production of interferon-gamma by T cells and NK cells and subsequently of the anti-angiogenic chemokines CXCL10/IP-10, and CXCL9/Mig. However the potent anti-tumor IL12 activity, which has been reported in mice, has not yet been successfully translated into clinical development, mainly because of early reports of severe toxicity and low response rates in human. Following our pioneering work which started in 2002 with the first description of an IL12 based antibody-cytokine fusion protein (i.e. Immunocytokine), we have extensively explored and perfected alternative molecular formats, with the aim to further improve biodistribution properties and therapeutic activity of IL12-based immunocytokines. Here we describe the development and evaluation of new targeted variants of both murine and human IL-12 with enhanced therapeutic efficacy and improved safety profile. To this end we combined the immunomodulatory properties of the IL12 payload with the tumor-homing activity of the L19 antibody. L19 is a clinical grade fully human antibody, which recognizes with identical affinity in both mouse and human, the alternatively spliced EDB domain of fibronectin. EDB represent an optimal target for anti-cancer pharmacodelivery, due to its pan-tumoral over-expression nature combined with very low expression levels in normal tissues. This has also been confirmed by extensive Nuclear Medicine work in which radiolabeled L19 has been administered to more than 150 patients, making L19 one of the best validated tumor-targeting agent. Our novel immunocytokine, termed IL12-L19-L19, relies on the L19 antibody in tandem diabody format, with a monomeric IL12 moiety expressed as single-chain polypeptide at the N-terminal extremity. Recombinant IL12-L19-L19 proteins based either on human or murine IL12, were efficiently expressed in CHO cells and purified to high quality. The tumor-targeting properties of both variants were validated in tumor-bearing mice, using radioiodinated protein preparations. In preclinical therapy studies IL12-L19-L19 showed potent anti-cancer activity when used either as single agent or in combination with other anticancer agents. PK studies in Cynomolgus Monkey using the fully human IL12-L19-IL19 product, revealed a favorable profile, which is compatible with other clinical-stage immunocytokines based on antibody-fragments. These results strongly support the further development of the fully human IL12-L19-L19 product for future clinical investigations. Citation Format: Mattia Matasci, Emanuele Puca, Tiziano Ongaro, Sarah Wulhfard, Alessandra Villa, Dario Neri. A novel immunocytokine for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5553.

Abstract 630: Novel hexavalent HVEM agonist HERA-LIGHT promotes T cell activation and expansion

Abstract The tumor necrosis factor superfamily (TNFSF) member LIGHT (TNFSF14) plays an important role in regulating the activity of immune cells, especially T cells. Several studies have shown that engagement of HVEM (herpesvirus entry mediator), one of the known receptors of LIGHT, can deliver a co-stimulatory signal to support T cell activation and expansion and promote tumor clearance. The HERA technology platform developed by Apogenix generates fully human hexavalent TNFSF fusion proteins that mimic the natural receptor binding mode in order to co-stimulate T cells. HERA ligands are pure agonists whose signaling capacity does not rely on secondary Fcγ-receptor crosslinking. Here we report the in vitro and in vivo properties of a novel HERA-LIGHT construct. Similar to all HERA fusion proteins, HERA-LIGHT has been engineered as a perfect molecular mimic of the natural ligand with high clustering capacity for the cognate receptor. The core unit consists of a single chain polypeptide comprising the three minimal LIGHT-subsequences necessary for folding into a functional trivalent receptor binding domain (RBD). By fusing a silenced IgG1 Fc-domain as a dimerization scaffold to the C-terminus of the RBD we generated HERA-LIGHT, a hexavalent fusion protein. HERA-LIGHT was expressed in CHO suspension cells followed by a lab-scale purification process including AFC- and SEC-based polishing, resulting in homogenous, aggregate-free protein lots. HERA-LIGHT was proven to bind both the human and murine HVEM receptor, as determined by ELISA. Qualitative analytics revealed excellent stability following heat- and pH-stress as well as freeze-thaw cycles. Analyzing serum samples from a PK study in CD1-mice, the terminal half-life of the compound was 36.5 hours. This short half-life, relative to antibodies, allows for fast-in/fast-out dynamics essential for improving combination therapy and reducing serious side effects associated with immune system overstimulation. In order to test biological activity, T cells were isolated by magnetic sorting from human PBMCs and treated with HERA-LIGHT in vitro. Flow cytometric analysis revealed that HERA-LIGHT enhanced activation and proliferation of naïve effector T cells (Teff) following stimulation with anti-CD3 antibody, as determined by CFSE dilution. Importantly, co-stimulation with HERA-LIGHT prevented regulatory T cell (Treg)-mediated suppression of Teff proliferation. In vivo, treatment with a murine surrogate of HERA-LIGHT resulted in significant tumor growth inhibition in a pilot study using the syngeneic CT-26 colorectal cancer model.In summary, the unique hexavalent design of HERA-LIGHT mediates efficient co-stimulation of Teff even in the presence of Treg cells and independent of secondary crosslinking events. Being true agonists, all HERA molecules are unique from current antibody-based concepts rendering them attractive candidates for cancer immunotherapy. Citation Format: Julian P. Sefrin, David M. Richards, Jaromir Sykora, Meinolf Thiemann, Christian Merz, Viola Marschall, Mauricio Redondo Müller, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. Novel hexavalent HVEM agonist HERA-LIGHT promotes T cell activation and expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 630.

Neuraminidase A from Streptococcus pneumoniae has a modular organization of catalytic and lectin domains separated by a flexible linker.

Neuraminidase A (NanA) of the pathogen Streptococcus pneumoniae cleaves receptors of the human respiratory epithelial surface during bacterial colonization. The full-size structure of NanA that contains one lectin and one catalytic domain within a single polypeptide chain remains unresolved. Both domains are crucial for the microorganism's virulence and considered as promising antimicrobial targets. Methods of bioinformatics and molecular dynamics have been implemented to model NanA's structure and study interaction between the lectin and catalytic domains in three neuraminidases NanA, NanB, and NanC from Streptococcus pneumoniae. A significant difference in spatial organization of these homologous enzymes has been revealed. The lectin and catalytic domains of NanB and NanC form rigid globules stabilized by multiple interdomain interactions, whereas in NanA, the two domains are separated by a 16 amino acids long flexible linker - a characteristic of proteins that require conformational flexibility for their functioning. The biological role of this structural adaptation of NanA as a key virulence enzyme is discussed.

A hypothesis explaining why so many pathogen virulence proteins are moonlighting proteins.

Moonlighting or multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Proteins that belong to key ancestral functions and metabolic pathways such as primary metabolism typically exhibit moonlighting phenomenon. We have collected 698 moonlighting proteins in MultitaskProtDB-II database. A survey shows that 25% of the proteins of the database correspond to moonlighting functions related to pathogens virulence activity. Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? Our hypothesis is that these proteins present a high conservation between the pathogen protein and the host counterparts. Therefore, the host immune system will not elicit protective antibodies against pathogen proteins. The fact of sharing epitopes with host proteins (known as epitope mimicry) might be the cause of autoimmune diseases. Although many pathogen proteins can be antigenic, only a few of them would elicit a protective immune response. This would also explain the lack of successful vaccines based in these conserved moonlighting proteins.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.