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PubMed2003

Antibodies as therapeutic agents: vive la renaissance!

Until recently, the concept of antibodies as in vivo therapeutics was still considered to be an exceedingly ambitious goal. However, in 2003, the situation has been completely transformed, with 14 FDA-approved monclonal antibodies (mAbs), 70 in late stage clinical (Phase II+) trials and > 1000 in preclinical development. The driving force behind this reversal in fortune has been advances in antibody engineering and the emergence of novel discovery techniques which overcame stability and immunogenicity issues that had blighted previous clinical trials of murine antibodies. For indications as diverse as inflammation, cancer and infectious disease, it is clear that unique properties of antibodies make them safe, effective and versatile therapeutics. These drugs can be used to neutralise pathogens, toxins and endogenous mediators of pathology. As cell targeting reagents, antibodies can be used to modulate cytoplasmic cascades or to 'tag' specific cells for complement- or effector-mediated lysis. Antibodies can also be modified to deliver toxic or modulatory payloads (small molecules, radionuclides and enzymes) and engineered to bind multiple epitopes (bispecifics) or even to have novel catalytic activity (abzymes). The modular structure of immunoglobulins and the availability of antibody fragment libraries also make it possible to produce variable-domain therapeutics (Fab, single-chain and domain antibodies). Although exhibiting less favourable kinetics in vivo, these fragments are simple to express and have an increased tissue penetration, making them especially useful as neutralising agents or in the delivery of payload. The number of approved antibodies is expected to increase arithmetically in the near term, as the platform is adopted as a valid alternative to small molecule discovery. This review provides an introduction to the antibody discovery process and discusses the past, present and future applications of therapeutic antibodies, with reference to several FDA-approved precedents.

Animals↗

PubMed2000

A Chimera Polypeptide with Active Sites of HWTX-I and AAI.

The functional amino acid sequence and its neighbouring fragments in the molecule of Amaranth alpha-amylase inhibitor isolated from seeds of the Mexican crop plant Amaranthus hypochondriacus were transferred, by solid phase chemical synthesis, into N-terminal region of the huwentoxin-I(HWTX-I). The synthetic chimera polypeptide was confirmed by Edman degradation and MALDI-TOF mass spectroscopy. The formation of three disulfide bonds and special conformation of the synthetic chimera was induced by the addition of glutathione. Renatured chimera polypeptide was purified by ion-exchange and reversed phase HPLC. The results showed that the engineered chimera polypeptide exerted obvious inhibitory activity to alpha-amylase from digestive tube of the roach (Periplaneta Americana) at pH 5.5 with the concentration of 9.5x10(-5) mol/L, and also exerted 36% of the neurotoxic activity of the natural huwentoxin-I as shown by the experiments of blockage of the neuromuscular transmission of isolated mouse phrenic nerve-diaphragm preparations. The experiments demonstrated that the structural motif of HWTX-I is well promising for protein engineering, and the solid-phase peptide synthesis is adequate rapid for the engineering of artificially designed small proteins.

Journal Article↗

PubMed1992

Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I.

The inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.

Amino Acid Sequence↗

PubMed2003

Control of peste des petits ruminants: classical and new generation vaccines.

Peste des Petits Ruminants is a highly contagious disease of domestic and wild small ruminants. It seems to be the major constraint in the development of small ruminant production in areas where it is endemic. Included in the list A of the International Zoosanitary Code, it is also part of the FAO EMPRESS programme. Classically, it is characterised by fever, nasal and ocular discharges, diarrhoea, respiratory distress, mucosal erosive lesions and death in 40-80% of acute cases. All these clinical signs, apart from the respiratory symptoms, are very similar to those of rinderpest. The causal agents of both diseases are closely related and belong to the Morbillivirus genus. In the absence of homologous vaccine, and taking advantage of the close relationship between the two viruses, the attenuated tissue culture rinderpest vaccine has been used for a long time to protect small ruminants against PPR. At the end of 1980s, a PPRV strain was successfully attenuated by serial passages in Vero cells. Demonstrated to be very efficient in the protection of sheep and goats against a virulent challenge, this avirulent PPRV is now widely used in the control of PPR. Its thermostability has been dramatically improved by freeze drying. However, animals inoculated with this vaccine cannot be distinguished serologically from those infected with the wild type viruses. With the reverse genetics technique, it is now possible to engineer PPR marker vaccines which can allow such a differentiation. The genes of the PPRV immune protective proteins, the fusion and the haemagglutinin proteins, have been introduced into the genome of a capripox vaccine strain. The generated viruses have proved to be effective as a dual vaccine to protect against two major diseases of small ruminants: peste des petits ruminants and capripox infections.

Animals↗

PubMed1995

Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus.

Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.

Animals↗

PubMed1998

Conformational changes necessary for gene regulation by Tet repressor assayed by reversible disulfide bond formation.

We constructed and characterized four Tet repressor (TetR) variants with engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by tetracycline (tc) might occur. All TetR mutants show nearly wild-type activities in vivo, and the reduced proteins also show wild-type activities in vitro. Complete and reversible disulfide bond formation was achieved in vitro for all four mutants. The disulfide bond in NC18RC94 immobilizes the DNA reading head with respect to the protein core and prevents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thus, these residues must have different conformations when bound to these ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobilize the variable loop between alpha-helices 8 and 9 located near the tc-binding pocket. A faster rate of disulfide formation in the operator-bound conformation and a lack of induction after disulfide formation show that the variable loop is located closer to the protein core in the operator-bound conformation and that a movement is necessary for induction. The disulfide bond in RC195VC199' connects alpha-helices 10 and 10' of the two subunits in the dimer and is only formed in the tc-bound conformation. The oxidized protein shows reduced operator binding. Thus, this bond prevents formation of the operator-bound conformation. The detection of conformational changes in three different regions is the first biochemical evidence for induction-associated global internal movements in TetR.

Cysteine↗

PubMed2006

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome.

Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses.

Animals↗

PubMed2003

Acellularized porcine heart valve scaffolds for heart valve tissue engineering and the risk of cross-species transmission of porcine endogenous retrovirus.

OBJECTIVE: Acellularized porcine heart valve scaffolds have been successfully used for heart valve tissue engineering, creating living functioning heart valve tissue. However, there is concern about the possibility of porcine endogenous retrovirus transmission. In this study we investigated whether acellularized porcine heart valve scaffold causes cross-species transmission of porcine endogenous retrovirus in a sheep model. METHODS: Acellularized porcine pulmonary valve conduits (n = 3) and in vitro autologous repopulated porcine pulmonary valve conduits (n = 5) were implanted into sheep in the pulmonary valve position. Surgery was carried out with cardiopulmonary bypass support. The animals were killed 6 months after the operation. Blood samples were collected regularly up to 6 months after the operation and tested for porcine endogenous retrovirus by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. In addition, explanted tissue-engineered heart valves were tested for porcine endogenous retrovirus after 6 month in vivo. RESULTS: Porcine endogenous retrovirus DNA was detectable in acellularized porcine heart valve tissue. However, 6 months after implantation of in vitro and in vivo repopulated acellularized porcine heart valve scaffolds, no porcine endogenous retrovirus sequences were detectable in heart valve tissue and peripheral blood. CONCLUSION: Acellularized porcine matrix scaffolds used for creation of tissue-engineered heart valves do not transmit porcine endogenous retrovirus.

Animals↗

PubMed2005

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket.

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.

Amino Acid Substitution↗

PubMed2005

Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells.

Human bone marrow (BM) mesenchymal stem/progenitor cells are potentially attractive targets for ex vivo gene therapy. The potential of lentiviral vectors for transducing BM mesenchymal cells was examined using a self-inactivating vector that expressed the green fluorescent protein (GFP) from an internal cytomegalovirus (CMV) promoter. This vector was compared with oncoretroviral vectors expressing GFP from the CMV promoter or a modified long-terminal repeat that had been optimized for long-term expression in stem cells. The percentage of GFP-positive cells was consistently higher following lentiviral versus oncoretroviral transduction, consistent with increased GFP mRNA levels and increased gene transfer efficiency measured by polymerase chain reaction and Southern blot analysis. In vitro GFP and FVIII expression lasted for several months post-transduction, although expression slowly declined. The transduced cells retained their stem/progenitor cell properties since they were still capable of differentiating along adipogenic and osteogenic lineages in vitro while maintaining high GFP and FVIII expression levels. Implantation of lentivirally transduced human BM mesenchymal cells using collagen scaffolds into immunodeficient mice resulted in efficient engraftment of gene-engineered cells and long-term transgene expression in vivo. These biocompatible BM mesenchymal implants represent a reversible, safe, and versatile protein delivery approach because they can be retrieved in the event of an unexpected adverse reaction or when expression of the protein of interest is no longer required. In conclusion, efficient gene delivery with lentiviral vectors in conjunction with the use of bioengineered reversible scaffolds improves the therapeutic prospects of this novel approach for gene therapy, protein delivery, or tissue engineering.

Adipogenesis↗

PubMed1985

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface.

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Numerical simulation of local pharmacokinetics of a drug after intravascular delivery with an eluting stent.

We use mathematical modelling to delineate the influence of two important factors on local pharmacokinetics of a drug delivered via an eluting stent, namely: (1) diffusional resistance of a stent coating, and (2) reversible binding of a drug to the vascular tissue. A system of differential equations that describes diffusion of the drug out of the polymeric coating of the stent into the vascular tissue and into the bloodstream, as well as reversible binding of the drug within the vascular tissue, was solved numerically and the spatial profiles of the concentration of the drug at various points of time were produced and analysed. Also, kinetic curves of the spatial average concentration of the drug within the wall were constructed, and the areas under those curves (AUC) were calculated. The simulations showed that AUC might be enhanced, if the stent is coated with a continuous layer of a drug-releasing medium with a high diffusional resistance. Both the residence time and the average concentration of the drug within the vascular wall increase in this case mainly because the coating imposes a diffusional barrier between the vascular tissue and the bloodstream, thereby reducing the wash-out. If the drug reversibly binds to the tissue, the residence time increases greatly, but the AUC for the free (unbound) drug remains unchanged, implying that the presence of the drug in the vessel is prolonged at the expense of a proportional reduction in concentration of a free drug within the tissue. These findings justify the design of eluting stents with continuous coatings with enhanced diffusional resistance and the engineering of drugs with enhanced affinity to the vascular matrix. Reversible binding to tissue may be beneficial for prolonging the presence of the drug in the target tissue, and for avoiding potential toxic peak effects of high concentrations of the free (unbound) drug.

Drug Delivery Systems↗

PubMed2003

Organ printing: computer-aided jet-based 3D tissue engineering.

Tissue engineering technology promises to solve the organ transplantation crisis. However, assembly of vascularized 3D soft organs remains a big challenge. Organ printing, which we define as computer-aided, jet-based 3D tissue-engineering of living human organs, offers a possible solution. Organ printing involves three sequential steps: pre-processing or development of "blueprints" for organs; processing or actual organ printing; and postprocessing or organ conditioning and accelerated organ maturation. A cell printer that can print gels, single cells and cell aggregates has been developed. Layer-by-layer sequentially placed and solidified thin layers of a thermo-reversible gel could serve as "printing paper". Combination of an engineering approach with the developmental biology concept of embryonic tissue fluidity enables the creation of a new rapid prototyping 3D organ printing technology, which will dramatically accelerate and optimize tissue and organ assembly.

Artificial Organs↗

PubMed2000

Site-directed ligand discovery.

We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

Amino Acid Substitution↗

PubMed2001

A unified theory of enzyme kinetics based upon the systematic analysis of the variations of k(cat), K(M), and k(cat)/K(M) and the relevant DeltaG(0 not equal) values-possible implications in chemotherapy and biotechnology.

To elucidate the kinetic properties of critical enzymatic situations that have previously escaped classification, we performed a systematic analysis of all the possible variations of the kinetic constants k(cat,) K(M,) and k(sp) = k(cat)/K(M,) encompassing all aspects of enzymology. The equation gives a total of thirteen theoretically possible cases, comprising the reference case plus 12 different sets of variations, which can be divided into six principal cases and six specular ones. The six relevant cases are examined individually in the context of each of the main chapters of enzymology, i.e. as regards mechanism of action, specificity of substrate and isoenzyme, reversible and irreversible inhibition, and mutation of residues (enzyme evolution and enzyme engineering). Some critical cases where k(sp) does not hold as a specificity index are classified for the first time. Interestingly, the six possible cases correspond to the five known cases of reversible inhibition (competitive, non-competitive, incompetitive, mixed competitive/non-competitive, and mixed incompetitive/non-competitive) plus an additional case of biphasic nature (activation-inhibition), which is crucial for a full understanding of specificity and which leads us to propose some modification to the definition of enzyme specificity. The systematic approach to enzymology outlined herein could find practical applications in various sectors of biotechnology, including chemotherapy.

Binding, Competitive↗

PubMed2005

Meeting product development challenges in manufacturing clinical grade oncolytic adenoviruses.

Oncolytic adenoviruses have been considered for use as anticancer therapy for decades, and numerous means of conferring tumor selectivity have been developed. As with any new therapy, the trip from the laboratory bench to the clinic has revealed a number of significant development hurdles. Viral therapies are subject to specific regulations and must meet a variety of well-defined criteria for purity, potency, stability, and product characterization prior to their use in the clinic. Published regulatory guidelines, although developed specifically for biotechnology-derived products, are applicable to the production of oncolytic adenoviruses and other cell-based products, and they should be consulted early during development. Most importantly, both the manufacturing process and the development of characterization and release assays should be science-driven, use the best available science and technology, and must consider the unique nature of the product: a living, and mutatable, virus. Potentially significant impacts on product quality and safety stem from the possibility of genetic instability related to over-engineering the viruses (as evidenced by their recombination and/or occasional reversion to wild-type virus during manufacturing). This report provides examples of some of the critical components affecting the development and production of clinical grade material and summarizes the significant progress made in recent years.

Adenoviridae↗

PubMed2001

Genetically engineered macrophages expressing IFN-gamma restore alveolar immune function in scid mice.

Reversal of immunodeficiency in the lung by gene therapy is limited in part by the difficulty of transfecting lung cells in vivo. Many options exist for successfully transfecting cells in vitro, but they are not easily adapted to the in vivo condition. To overcome this limitation, we transduced macrophages in vitro with the murine IFN-gamma (mIFN-gamma) gene and intratracheally delivered the macrophages to express mIFN-gamma in vivo. A recombinant retroviral vector pSF91 system was modified to encode mIFN-gamma and enhanced green fluorescent protein (EGFP). A murine macrophage cell line J774A.1 transduced with the retroviral supernatant increased secretion from undetectable levels to 131.6 +/- 4.2 microg/ml mIFN-gamma at 24 h in vitro. The mIFN-gamma-producing macrophages were intratracheally instilled into mechanically ventilated scid mice. mIFN-gamma levels in the bronchoalveolar lavage increased from undetectable levels at baseline to 158.8 +/- 5.1 pg/ml at 48 h (P < 0.001). Analysis of the lavaged cells for EGFP expression revealed that EGFP expression was directly proportional to the number of transduced macrophages instilled into the lung. Immune function was partially restored in the alveolar spaces of scid mice with evidence of enhanced MHC class II antigen expression and increased phagocytosis (P < 0.05). Tumor necrosis factor alpha was increased from undetectable at baseline to 103.5 +/- 11.4 pg/ml. In contrast, i.p. administration of the engineered macrophages did not enhance IFN-gamma levels in the lung. Our study suggests airway delivery of genetically engineered macrophages expressing mIFN-gamma gene can partially restore significant immune activity in the lungs of immunodeficient mice.

Animals↗

PubMed2006

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.

Base Sequence↗