scholarly journals Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins

2000 ◽  
Vol 345 (2) ◽  
pp. 247-254 ◽  
Author(s):  
Anita GOYAL ◽  
Janendra K. BATRA
Keyword(s):  
Therapeutic Potential ◽  
Cell Killing ◽  
Cytotoxic Activities ◽  
Single Chain ◽  
Killing Activity ◽  
C Terminus ◽  
Human Transferrin Receptor ◽  
N Terminus ◽  
Target Cell Line

Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. A 12-residue spacer, Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu, containing the recognition site for the protease furin, was incorporated between the toxin and the ligand to generate restrictocin-spacer-anti-TFR(scFv) and anti-TFR(scFv)-spacer-restrictocin. The incorporation of the proteolytically cleavable spacer enhanced the cell-killing activity of both constructs by 2-30-fold depending on the target cell line. However, the introduction of linker improved the cytotoxic activity only for anti-TFR(scFv)-linker-restrictocin. The proteolytically cleavable spacer-containing chimaeric toxins had similar cytotoxic activities irrespective of the location of the ligand on the toxin and they were found to release the restrictocin fragment efficiently on proteolysis in vitro.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

Mimicking cotranslational folding of prosubtilisin E in vitro

2020 ◽  
Vol 167 (5) ◽  
pp. 473-482 ◽  
Author(s):  
Sung-Gun Kim ◽  
Yu-Jen Chen ◽  
Liliana Falzon ◽  
Jean Baum ◽  
Masayori Inouye
Keyword(s):  
Crucial Role ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Secondary Structures ◽  
Terminal Region ◽  
Folding Intermediates ◽  
C Terminus ◽  
Cotranslational Folding ◽  
N Terminus

Abstract Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

scholarly journals Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Yoko Usami ◽  
Yukihiro Kobayashi ◽  
Takahiro Kameda ◽  
Akari Miyazaki ◽  
Kazuyuki Matsuda ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Cleavage Sites ◽  
Carboxypeptidase A ◽  
Apolipoprotein A ◽  
C Terminus ◽  
N Terminus ◽  
Plaque Destabilization ◽  
New Biomarkers ◽  
A Minor

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe225) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL3) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe229 and Tyr192 residues were the main cleavage sites. Interestingly, the Phe225 residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL3; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL3 at only the N-terminus, especially at Phe33. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe225 and Phe229 residues newly exposed by chymase, but did not cleave Tyr192. These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).

Targeting MHC-linked wild type p53 with TCR mimic single chain diabody for cancer immunotherapy.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2524-2524
Author(s):  
Suman Paul ◽  
Jacqueline Douglass ◽  
Annika Schaefer ◽  
Emily Han-Chung Hsiue ◽  
Alexander Pearlman ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cancer Immunotherapy ◽  
Cell Killing ◽  
Human Cells ◽  
Wild Type ◽  
Single Chain ◽  
Nsg Mice ◽  
Wild Type P53

2524 Background: Increased tumor suppressor protein p53 expression is observed in a wide range of human cancers. As a result there is intense interest in targeting p53 for cancer therapy. Intracellular p53 is inaccessible to therapeutic antibodies that bind cell surface proteins. However, intracellular proteins including p53 are degraded into peptides that are presented on cell surface in association with HLA class I molecules. Thus p53 peptide-HLA (p53-HLA) complexes can be antibody targets. Methods: Using phage display we identified a novel anti-p53-HLA single chain variable fragment (scFv) clone-43 that recognizes a wild-type p53 10-mer epitope bound to HLA-A*2402. By coupling our clone-43 scFv with an anti-CD3 scFv, we generated a single chain diabody (scDb) designed to activate T-cells against p53-expressing target cells. Results: In-vitro co-culture of clone-43 scDb with donor human T-cells and p53 expressing SIG-M5 cancer cells results in SIG-M5 cell killing and concomitant T-cell interferon gamma (IFNγ) release. In contrast, similar co-culture with SIG-M5 p53-knock out (KO) cells showed no cell killing and minimal IFNγ release demonstrating specificity of clone-43 to p53 expressing cells. Additionally, in-vivo growth of p53 expressing SW480 cancer cell xenografts in NSG mice was completely terminated by clone-43 scDb injections. A major concern for wild-type p53 epitope targeting is potential on-target off-tumor effect on non-cancerous tissue. We observed significant in-vitro clone-43 scDb mediated killing of human HLA-A*24:02 peripheral blood mononuclear cells. To better evaluate effect of clone-43 scDb on non-neoplastic human cells, we engrafted HLA-A*24:02 human CD34+ hematopoietic stem cells into NSG mice to generate a humanized mouse model with circulating mature human CD45+ cells. Clone-43 scDb treatment resulted in selective depletion of circulating human cells while the same cells persisted in mice treated with unrelated control scDb. Conclusions: Our observation that immune targeting of wild-type p53 epitope results in significant off-tumor hematopoietic cell death is contrary to previously published reports and carries important implications for future anti-p53 antibody and vaccine design for cancer immunotherapy.

scholarly journals Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

2000 ◽  
Vol 182 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
Volkmar Braun
Keyword(s):  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Binding Assays ◽  
Surface Binding ◽  
Citrate Transport ◽  
C Terminus ◽  
N Terminus ◽  
Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

scholarly journals Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK inSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2425-2440 ◽  
Author(s):  
Cunle Wu ◽  
Ekkehard Leberer ◽  
David Y. Thomas ◽  
Malcolm Whiteway
Keyword(s):  
Protein Kinase ◽  
Functional Characterization ◽  
Hog Pathway ◽  
C Terminus ◽  
Extracellular Signal ◽  
Osmotic Stress Response ◽  
N Terminus

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

scholarly journals Effects of l- and d-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication

2002 ◽  
Vol 366 (3) ◽  
pp. 863-872 ◽  
Author(s):  
Bouchaib BAHBOUHI ◽  
Nathalie CHAZAL ◽  
Nabil Georges SEIDAH ◽  
Cristina CHIVA ◽  
Marcelo KOGAN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Envelope Glycoprotein ◽  
Cleavage Site ◽  
Molecular Level ◽  
Principal Cell ◽  
Inhibition Constant ◽  
Glycoprotein Precursor ◽  
C Terminus ◽  
N Terminus

The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1BRU gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35μM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70μM. The data show that only dec14d and dec14dcmk could inhibit HIV-1BRU, HIV-2ROD and SIVmac251 replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant Ki = 4.6μM, whereas the peptide dec14l preferentially inhibited furin with a Ki = 28μM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors.

Actopaxin is phosphorylated during mitosis and is a substrate for cyclin B1/cdc2 kinase

2002 ◽  
Vol 363 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Michael CURTIS ◽  
Sotiris N. NIKOLOPOULOS ◽  
Christopher E. TURNER
Keyword(s):  
Cell Division ◽  
Focal Adhesions ◽  
Cyclin B1 ◽  
Actin Binding ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
N Terminus ◽  
Focal Adhesion Protein

Prior to cell division, normal adherent cells adopt a round morphology that is associated with a loss of actin stress fibres and disassembly of focal adhesions. In this study, we investigate the mitotic phosphorylation of the recently described paxillin and actin-binding focal-adhesion protein actopaxin [Nikolopoulos and Turner (2000) J. Cell Biol. 151, 1435–1448]. Actopaxin is comprised of an N-terminus containing six putative cdc2 phosphorylation sites and a C-terminus consisting of tandem calponin homology domains. Here we show that the N-terminus of actopaxin is phosphorylated by cyclin B1/cdc2 kinase in vitro and that this region of actopaxin precipitates cdc2 kinase activity from mitotic lysates. Actopaxin exhibits reduced electrophoretic mobility during mitosis that is dependent on phosphorylation within the first two consensus cdc2 phosphorylation sites. Finally, as cells progress from mitosis to G1 there is an adhesion-independent dephosphorylation of actopaxin, suggesting that actopaxin dephosphorylation precedes cell spreading and the reformation of focal adhesions. Taken together, these results suggest a role for cyclin B1/cdc2-dependent phosphorylation of actopaxin in regulating actin cytoskeleton reorganization during cell division.

scholarly journals Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action

1997 ◽  
Vol 324 (3) ◽  
pp. 815-822 ◽  
Author(s):  
Dharmendar RATHORE ◽  
Janendra K. BATRA
Keyword(s):  
Transferrin Receptor ◽  
Metabolic Inhibitors ◽  
Target Cells ◽  
Cytotoxic Activities ◽  
Dna Encoding ◽  
Single Chain ◽  
Human Transferrin ◽  
Human Transferrin Receptor ◽  
A Cell

Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)–restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5′ end of that encoding restrictocin. The other chimaeric toxin, restrictocin–anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3′ end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitrotranslation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin–anti-TFR(scFv) was more active than anti-TFR(scFv)–restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus.

CAP-100: First-in-class antibody for CCR7+ hematological malignancies.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e19008-e19008
Author(s):  
Carlos Cuesta ◽  
Cecilia Munoz-Callega ◽  
Javier Loscertales ◽  
Fernando Terron ◽  
Wim Mol
Keyword(s):  
T Cell ◽  
B Cell ◽  
Hematological Malignancies ◽  
Surface Expression ◽  
Cell Killing ◽  
Tumor Growth Inhibition ◽  
Killing Activity ◽  
T Lymphomas

e19008 Background: CCR7 is highly expressed in many hematological malignancies including CLL, several B-cell non-Hodgkin lymphomas (NHL), and various T-cell neoplasias with nodal involvement. Upon engagement by its ligands (CCL19 and CCL21), CCR7 controls trafficking of cells to locations where these chemokines are expressed, such as the lymph node (LN) and central nervous system. In these protective microenvironments CCR7 ligands contribute to tumor cell survival and proliferation. Indeed, both high CCR7 surface expression levels and high migratory responses to CCR7 ligands correlate with LN involvement, adverse prognostic factors, and shorter patient survival. Thus, strategies targeting CCR7 could provide a novel therapeutic approach for CCR7+ hematological malignancies. Methods: We have generated CAP-100, the first humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand-mediated signaling from both CCL19 and CCL21, and evaluated the antibody in various in-vitro and in-vivo preclinical models. Results: CAP-100 effectively inhibited in vitro migration of primary patient samples of CLL, B-cell NHLs and T-cell neoplasias such as T-PLL or T-ALL. Furthermore, in in vivo pre-clinical studies, CAP-100 was shown to inhibit entry of CCR7-expressing cells to LNs. CAP-100 also abrogated survival elicited by CCR7 in CLL, and showed potent cell killing activity against CLL or CCR7+ T-lymphomas cells. This Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) clearly outperformed anti-CD20 or anti-CD52 standard-of-care antibodies in B-NHL and T-lymphomas respectively. In all cases, ADCC and migration inhibition were both independent of prognostic markers for high risk disease. Finally, when given as monotherapy in disseminated B-NHL and CLL xenograft tumors in SCID mice, CAP-100 exhibited tumor growth inhibition and extended survival significantly. Conclusions: Our results demonstrate that CAP-100, the first-in-class anti-CCR7 mAb, is a potent antagonist with biological activity in several CCR7+ hematological malignancies, including relapsed/refractory disease. Moreover, these results highlight the relevance of the CCR7-CCL19/CCL21 pathway as a therapeutic target in these diseases. CAP-100’s unique propensity to block migration of tumor cells to the LN, in combination with its potent cell killing activity provides the biological rationale for use of CAP-100, either as monotherapy or in combination with novel agents. Clinical trials in CLL and CCR7-expressing NHL will be initiated soon.

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