Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity

The interdomain instability of single-chain fragment variable (scFv) might result in intermolecular aggregation and loss of function. In the present study, we stabilized H4-an anti-aflatoxin B(1) (AFB(1)) scFv-with an interdomain disulfide bond and studied the effect of the disulfide bond on antibody affinity. With homology modeling and molecular docking, we designed a scFv containing an interdomain disulfide bond between the residues H44 and L100. The stability of scFv (H4) increased from a GdnHCl(50) of 2.4 M to 4.2 M after addition of the H44-L100 disulfide bond. Size exclusion chromatography revealed that the scFv (H44-L100) mutant existed primarily as a monomer, and no aggregates were detected. An affinity assay indicated that scFv (H4) and the scFv (H44-L100) mutant had similar IC(50) values and affinity to AFB(1). Our results indicate that interdomain disulfide bonds could stabilize scFv without affecting affinity.     

Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C

An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.     

The Ability of Chlorophyll to Trap Carcinogen Aflatoxin B1: A Theoretical Approach

The coordination of one and two aflatoxin B₁ (AFB₁, a potent carcinogen) molecules with chlorophyll a (chl a) was studied at a theoretical level. Calculations were performed using the M06-2X method in conjunction with the 6-311G(d,p) basis set, in both gas and water phases. The molecular electrostatic potential map shows the chemical activity of various sites of the AFB₁ and chl a molecules. The energy difference between molecular orbitals of AFB₁ and chl a allowed for the establishment of an intermolecular interaction. A charge transfer from AFB₁ to the central cation of chl a was shown. The energies of the optimized structures for chl a show two configurations, unfolded and folded, with a difference of 15.41 kcal/mol. Chl a appeared axially coordinated to the plane (α-down or β-up) of the porphyrin moiety, either with the oxygen atom of the ketonic group, or with the oxygen atom of the lactone moiety of AFB₁. The complexes of maximum stability were chl a 1-α-E-AFB₁ and chl a 2-β-E-AFB₁, at −36.4 and −39.2 kcal/mol, respectively. Additionally, with two AFB₁ molecules were chl a 1-D-2AFB₁ and chl a 2-E-2AFB₁, at −60.0 and −64.8 kcal/mol, respectively. Finally, biosorbents containing chlorophyll could improve AFB₁ adsorption.     

Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V_H–V_L sequence with a (Gly₄Ser)₃ linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻⁴ M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 10⁷ M⁻¹ (k_on/k_off), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.     

Effects of Aflatoxin B1 on T-Cell Subsets and mRNA Expression of Cytokines in the Intestine of Broilers

This study was conducted to investigate the effects of aflatoxin B₁ (AFB₁) on T-cell subsets and mRNA expression of cytokines in the small intestine of broilers. One hundred and fifty-six one-day-old healthy Cobb broilers were randomly divided into control group (0 mg/kg AFB₁) and AFB₁ group (0.6 mg/kg AFB₁) with three replicates per group and 26 birds per replicate for 21 days, respectively. At 7, 14, and 21 days of age, the duodenum, jejunum and ileum were sampled for analyzing T cell subsets (CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺) by flow cytometry as well as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α mRNA expression by qRT-PCR. The percentages of T-cells in the intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of duodenum, jejunum and ileum in the AFB₁ group showed a decreased tendency in comparison to the control group. The mRNA expression of cytokines in the three intestinal segments in the AFB₁ group presented a general decline compared with the control groups. Our data demonstrated that 0.6 mg/kg AFB₁ in the broilers diet could reduce the percentages of T-cell subsets and the expression level of cytokine mRNA in the small intestine, implying that the immune function of the intestinal mucosa might be affected. The reduction of cytokines mRNA expression may be closely associated with the decreased proportions of T cells subsets induced by AFB₁.     

Disulfide stabilization of antibody Fv: computer predictions and experimental evaluation

Using molecular modeling technology we have recently identifiedpositions in conserved framework regions of Fvs which can beused to stabilize antibody Fvs by an interchain disulfide bondengineered in between the structurally conserved framework positionsof the variable domains of heavy (V_H) and light (V_L) immunoglobulinchains (disulfide-stabilized Fv; dsFv). The computer model indicatedthe existence of other potential sites in the framework regionsthat might be suitable for disulfide bond formation betweenV_H and V_L. The possibility of obtaining dsFvs using these positionsis evaluated here experimentally by constructing dsFv immunotoxinsin which the Fv moiety is fused to a truncated form of Pseudomonasexotoxin. We analyzed the extent of dsFv formation and the activityof the resulting dsFv immunotoxins, and compared various dsFvmolecules with the scFv immunotoxin. Our results demonstratethat position H44-L105 is the only one which gives high productionyields of active dsFv. All other positions gave either low yieldsand activity or completely failed to produce active dsFv. Withone exception, the formation and activities of the dsFvs correspondedto the C-C distance between the VH and VL positions, with anoptimal distance of 5.7 Å producing the best dsFv. Distancesof 6.0–6.9 Å resulted in a' low yield of proteinthat was still capable of binding antigen, whereas distances>7.0 Å resulted in molecules in which dsFv formationwas not obtained.     

Highly unsaturated binuclear butadiene iron carbonyls: quintet spin States, perpendicular structures, agostic hydrogen atoms, and iron-iron multiple bonds

The highly unsaturated binuclear butadiene iron carbonyls (C₄H₆)₂Fe₂(CO)_n (n = 2, 1) have been examined using density functional theory. For (C₄H₆)₂Fe₂(CO)_n (n = 2, 1), both coaxial and perpendicular structures are found. The global minima of (C₄H₆)₂Fe₂(CO)_n (n = 2, 1) are the perpendicular structures 2Q-1 and 1Q-1, respectively, with 17- and 15-electron configurations for the iron atoms leading to quintet spin states. The Fe=Fe distance of 2.361 Å (M06-L) in the (C₄H₆)₂Fe₂(CO)₂ structure 2Q-1 suggests a formal double bond. The Fe≡Fe bond distance in the (C₄H₆)₂Fe₂(CO) structure 1Q-1 is even shorter at 2.273 Å (M06-L), suggesting a triple bond. Higher energy (C₄H₆)₂Fe₂(CO)_n (n = 2, 1) structures include structures in which a bridging butadiene ligand is bonded to one of the iron atoms as a tetrahapto ligand and to the other iron atom through two agostic hydrogen atoms from the end CH₂ groups. Singlet (C₄H₆)₂Fe₂(CO) structures with formal Fe–Fe quadruple bonds of lengths ∼2.05 Å were also found but at very high energies (∼47 kcal/mol) relative to the global minimum.     

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.     

Towards a universal disulphide stabilised single chain Fv format: importance of interchain disulphide bond location and vL-vH orientation

Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. Five of these disulphide bond locations were introduced into one scFv in order to compare their relative effects on expression, thermal stability, percent monomer formation and retention of antigen binding. The disulphide bond position vH44-vL100 was observed to enable the most favourable balance of biophysical properties. The vH44-vL100 disulphide bond was introduced into five additional scFv in both vL-vH and vH-vL orientations in order to investigate its general applicability. Data are presented to show the relative influence of scFv sequence, v-region organisation and interchain disulphide bond on expression yield, thermal stability and percent monomer. Introduction of the vH44-vL100 disulphide bond typically resulted in no or little increase in thermal stability and no change in percent monomer but did confer the benefit of permanently fixing monomer:dimer ratios during purification and analysis.     

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero- residue linker a trimer

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five- residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross- link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.      

Concerning the Radical Character of Superoxide. The H—O Bond Energy of HO−2

The strength of the H-O bond in H⁻O₂ should be a good indicator of the chemical reactivity of superoxide, O⁻₂, as a hydrogen atom abstractor. This quantity can be calculated if the electron affinity of HO₂ is known. A recent experimental determination of the electron affinity of HO₂ [V. M. Bierbaum, R. J. Schmitt and C. H. DePuy, J. Am. Chem. Soc., 103 , 6262–6263 (1981)] gave that value as 27.4 ± 0.2 kcal mol⁻¹. The H-O bond energy of HO⁻⁻₂ can then be calculated from that value to be approximately 66 kcal mol⁻¹. Various theoretical methods may also be used to approximate the value of the electron affinity of HO₂. These methods generally give values in accord with the experimental result. Based on the low H—O bond energy of 66 kcal mol⁻¹ for HO⁻₂, superoxide is expected to be relatively unreactive, comparable to iodine atoms but less reactive than bromine atoms or HO₂ and much less reactive than chlorine atoms or hydroxyl radicals. Based on our analysis, we predict that superoxide can only react as a hydrogen atom abstractor with substrates that contain relatively weak bonds to hydrogen such as hydroxylamine or hydroquinones.     

Anthracene-Walled Acyclic CB[n] Receptors: in vitro and in vivo Binding Properties toward Drugs of Abuse

We report studies of the interaction of six acyclic CB[n]-type receptors toward a panel of drugs of abuse by a combination of isothermal titration calorimetry and ¹H NMR spectroscopy. Anthracene walled acyclic CB[n] host ( M3 ) displays highest binding affinity toward methamphetamine (K_d=15 nM) and fentanyl (K_d=4 nM). Host M3 is well tolerated by Hep G2 and HEK 293 cells up to 100 μM according to MTS metabolic and adenylate kinase release assays. An in vivo maximum tolerated dose study with Swiss Webster mice showed no adverse effects at the highest dose studied (44.7 mg kg⁻¹). Host M3 is not mutagenic based on the Ames fluctuation test and does not inhibit the hERG ion channel. In vivo efficacy studies showed that pretreatment of mice with M3 significantly reduces the hyperlocomotion after treatment with methamphetamine, but M3 does not function similarly when administered 30 seconds after methamphetamine.     

Engineering a CD123xCD3 bispecific scFv immunofusion for the treatment of leukemia and elimination of leukemia stem cells

Engineered bispecific antibodies that recruit cytotoxic lymphocytes to kill specific tumor cells have been showing promising clinical results. Here, we describe a bispecific single-chain Fv (scFv) immunofusion or BIf to target CD123(+) leukemia, that contains an anti-CD123 scFv fused at the N-terminus of human IgG1 hinge-C(H)2-C(H)3, and an anti-CD3 scFv fused at C-terminus. When expressed from transfected CHO-S cells, CD123xCD3 BIf forms a homodimer that provides a structure of N-terminal tumor-targeting domain that closely resembles natural antibody. The CD123xCD3 dimeric structure also provides binding affinity to CD123(+) tumor cells with a Kd of 10(-10) M, one to two orders of magnitude stronger than traditional bispecific antibody constructs. The location of the anti-CD3 scFv at C-terminus of BIf reduces the binding affinity to CD3(+) T cells by two orders, which could help to prevent non-specific T-cell activation. CD123xCD3 BIf is able to achieve T-cell-mediated target cell killing activities at low pM levels with E/T ratios as low as 2. Overall, the inclusion of human IgG1 constant region in BIf construct increases target cell-binding affinity; potentially increases serum half-life by its larger size and FcRn-mediated salvage system; and includes the abilities to activate the additional antibody-mediated cellular cytotoxicities.     

A potential antitumor drug (arginine deiminase) reengineered for efficient operation under physiological conditions

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas, and studies on human lymphatic leukemia cell lines have confirmed that ADI has antiangiogenic activity. Recent studies showed that a combination of taxane and ADI‐PEG20, which induces caspase‐independent apoptosis, is more effective than taxane monotherapy for prostate cancer. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) and of many other ADI enzymes lies in their pH‐dependent activity profile. PpADI has a pH optimum at 6.5 and a pH shift from 6.5 to 7.5 results in an ～80 % activity drop (the pH of human plasma is 7.35 to 7.45). In 2010, we reported a proof of concept for ADI engineering by directed evolution that resulted in variant M2 (K5T/D44E/H404R). M2 has a pH optimum of pH 7.0, a fourfold higher k_cat value than the wild‐type PpADI (pH 7.4, 0.5 M phosphate buffer), and an increased K_m value for substrate arginine. In our latest work, variants M5 (K5T/D38H/D44E/A128T/H404R) and M6 (K5T/D38H/D44E/A128T/E296K/H404R) were generated by directed evolution by employing PBS buffer (pH 7.4), which mimics physiological conditions. The S_0.5 value of parent M3 (K5T/D44E/A128T/H404R) decreased from 2.01 to 1.48 mM (M5) and 0.81 mM (M6). The S_0.5 value of M6 (0.81 mM ) is lower than that of wild‐type PpADI (1.30 mM ); the k_cat values improved from 0.18 s^?1 (wild‐type PpADI) to 17.56 s^?1 (M5, 97.6‐fold) and 11.64 s^?1 (M6, 64.7‐fold).     

Reaction mechanism and kinetics for hydrolytic dehydrogenation of ammonia borane on a Pt/CNT catalyst

A reaction mechanism is proposed for hydrolytic dehydrogenation of ammonia borane on a Pt/CNT catalyst. A combination of thermodynamic analysis and FTIR measurement reveals that B‐containing byproducts are mainly in the form of an NH₄B(OH)₄‐B(OH)₃ mixture rather than NH₄BO₂ reported previously. The revised main reaction is , involving the B–H, B–N, and O–H bond cleavages. Isotopic experiments using D₂O instead of H₂O as reactant or introducing D₂ into the reaction atmosphere suggest the O–H bond cleavage being in the rate‐determining step, and an unfavorable occurrence of the chemisorbed H₂O dissociation (i.e., the direct O–H bond cleavage), respectively. Different reaction pathways with indirect O–H bond cleavages are analyzed, and then is suggested as the rate‐determining step. Subsequently, a Langmuir–Hinshelwood kinetic model is developed, which fits well with the experimental data. © 2016 American Institute of Chemical Engineers AIChE J, 63: 60–65, 2017     

Palladium (II) and platinum (II) complexes containing organometallic thiosemicarbazone ligands: Synthesis,characterization, X-ray structures and antitubercular evaluation

A new series of palladium (II) and platinum (II) complexes containing ferrocenyl and cyrhetrenyl thiosemicarbazone ligands were synthesized and characterized. The two-step reaction of the organometallic thiosemicarbazones with i) K₂MCl₄ and ii) PPh₃ and their subsequent recrystallization from CH₂Cl₂/hexane yielded the binuclear complexes [Mˋ{ML_n(η⁵-C₅H₄)C(H)NNC(S)NHR}–(Cl)(PPh₃)] (M′Pd, Pt; ML_nRe(CO)₃, FeCp; RH, CH₃). The structures of the products were inferred from elemental analyses and IR, ¹H and ³¹P NMR spectroscopies. The molecular structures of 2b and 3d were confirmed by single crystal X-ray analysis. All complexes were screened in vitro against Mycobacterium tuberculosis and exhibited only moderate activity in the low micromolar range.     

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (T_m,app, 72.5 °C) and an over 200‐fold longer half‐life at 60 °C. The most stable LinB variants also displayed increased compatibility with co‐solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild‐type enzyme.     

A form of anti-Tac(Fv) which is both single-chain and disulfide stabilized: comparison with its single-chain and disulfide-stabilized homologs

Disulfide-stabilized Fvs (dsFvs) are recombinant proteins composed of a heavy-chain variable domain (VH) of an antibody connected via a disulfide bond to the light-chain variable domain (VL). In single-chain Fvs (scFvs), a peptide connector links VH and VL. The dsFv form of the anti-Tac monoclonal antibody which reacts with the alpha subunit of the IL2 receptor was recently reported to be more stable and to aggregate less during renaturation than anti-Tac(scFv). In addition, it could be produced in a better yield owing to less aggregation. However, the yields are still too low to permit the production of material for clinical trials in which the dsFv will be used to image or treat IL2 receptor (CD25)-containing tumors. To increase the efficiency by which VH and VL associate and form a disulfide bond during renaturation, we have prepared an Fv form of anti-Tac which is both single chain and disulfide stabilized (scdsFv). The recombinant protein is expressed in Escherichia coli, where it accumulates in inclusion bodies. Using inclusion body protein as the reference point, the yield of purified anti-Tac(scdsFv) was 13% compared with 2% for anti-Tac(dsFv). Anti- Tac(scdsFv) has equivalent binding affinity, immunoreactivity after radiolabeling and stability. The results show that a linker between VH and VL facilitates heterodimer formation and leads to disulfide bond formation in a higher percentage of the molecules renatured. Thus anti- Tac(scdsFv) is the preferred form of anti-Tac(Fv) to be used for clinical studies. We anticipate that scdsFvs will be the optimum recombinant form of Fv to produce from bacteria.      

Mycoflora and Co-Occurrence of Fumonisins and Aflatoxins in Freshly Harvested Corn in Different Regions of Brazil

Natural mycoflora and co-occurrence of fumonisins (FB₁, FB₂) and aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂) in freshly harvested corn grain samples from four regions of Brazil were investigated. Fusarium verticillioides was predominant in all samples. Analysis of fumonisins showed that 98% of the samples were contaminated with FB₁ and 74.5% with FB₁ + FB₂, with toxin levels ranging from 0.015 to 9.67 μg/g for FB₁ and from 0.015 to 3.16 μg/g for FB₂. Twenty-one (10.5%) samples were contaminated with AFB₁, seven (3.5%) with AFB₂ and only one (0.5%) with AFG₁ and AFG₂ Co-contamination with aflatoxins and fumonisins was observed in 7% of the samples. The highest contamination of fumonisins and aflatoxins was observed in Nova Odessa (SP) and Várzea Grande (MT), respectively. The lowest contamination of these mycotoxins was found in Várzea Grande and Nova Odessa, respectively.     

Unsymmetrical cyrhetrenyl and ferrocenyl azines derived from 5-nitrofurane: Synthesis,structural characterization and electrochemistry

A new series of unsymmetrical cyrhetrenyl and ferrocenyl azines that were monosubstituted [(η⁵-C₅H₄)–C(R)N–NCH(5-NO₂–2-C₄H₂O)]M {with MRe(CO)₃ and RH (1a) or RMe (1b); MFe(η⁵-C₅H₅) and RH (2a) or RMe (2b)} and disubstituted [Fe{(η⁵-C₅H₄)–C(Me)N–NCH(5-NO₂–2-C₄H₂O)}₂] (3a) were prepared by condensation reactions of the corresponding organometallic hydrazone [(η⁵-C₅H₄)–C(R)N–NH₂)]M with 5-nitro-2-furaldehyde. The ¹H and ¹³C{¹H} NMR spectra indicated that these compounds adopted an (E,E)-configuration about the ˃CN − bond and an s-trans conformation about the N1–N2 bond, and this result was confirmed by X-ray crystallographic analyses of 1a and 2b. The opposite electronic effects of the organometallic fragments correlate with the co-planarity of the [(η⁵-C₅H₄)–C(R)N–NCH(5-NO₂–2-C₄H₂O)] system, the reduction potential of the nitro group (E_1/2) and the chemical shifts of the iminic carbons.