Abstract: Protein splicing in trans by split inteins has increasingly become a powerful protein-engineering tool for protein ligation, both in vivo and in vitro. Over 100 naturally occurring and artificially engineered split inteins have been reported for protein ligation using protein trans-splicing. Here, we review the current status of the reported split inteins in order to delineate an empirical or rational strategy for constructing new split inteins suitable for various applications in biotechnology and chemical biology.

Abstract: Protein engineering remains an area of growing importance in pharmaceutical and biotechnology research. Stabilization of a folded protein conformation is a frequent goal in projects that deal with affinity optimization, enzyme design, protein construct design, and reducing the size of functional proteins. Indeed, it can be desirable to assess and improve protein stability in order to avoid liabilities such as aggregation, degradation, and immunogenic response that may arise during development. One way to stabilize a protein is through the introduction of disulfide bonds. Here, we describe a method to predict pairs of protein residues that can be mutated to form a disulfide bond. We combine a physics-based approach that incorporates implicit solvent molecular mechanics with a knowledge-based approach. We first assign relative weights to the terms that comprise our scoring function using a genetic algorithm applied to a set of 75 wild-type structures that each contains a disulfide bond. The method is then tested on a separate set of 13 engineered proteins comprising 15 artificial stabilizing disulfides introduced via site-directed mutagenesis. We find that the native disulfide in the wild-type proteins is scored well, on average (within the top 6% of the reasonable pairs of residues that could form a disulfide bond) while 6 out of the 15 artificial stabilizing disulfides scored within the top 13% of ranked predictions. Overall, this suggests that the physics-based approach presented here can be useful for triaging possible pairs of mutations for disulfide bond formation to improve protein stability.

Abstract: A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.

Abstract: Therapeutic antibodies need to be highly resistant to aggregation due to the high concentrations required for subcutaneous delivery and the potential immunogenicity of antibody aggregates. Human antibody fragments-such as single-domain antibodies (VH or VL)-are typically much less soluble than full-length antibodies. Nevertheless, some aggregation-resistant VH domains have been discovered that are negatively charged at neutral pH and/or enriched in negatively charged residues within the complementarity-determining regions (CDRs). To better understand how to engineer diverse domain antibodies to resist aggregation, we have investigated the solubilizing activity of positively and negatively charged mutations within hydrophobic CDRs of multiple VH scaffolds that differ in their net charge. We find that negatively charged mutations inserted near the edges of hydrophobic CDRs are more effective than positively charged ones at inhibiting aggregation for VH scaffolds that are negatively or near-neutrally charged. In contrast, positively charged CDR mutations prevent aggregation better than negatively charged ones for a VH scaffold that is highly positively charged. Our findings suggest that the net charge of the antibody scaffold is a key determinant of the optimal CDR mutations for preventing aggregation. We expect that our findings will improve the design of aggregation-resistant antibodies with single- and multidomain scaffolds.

Abstract: Single domain antibodies, recombinant variable heavy domains derived from the unique heavy-chain only antibodies found in camelids and sharks, are exceptionally rugged due to their ability to refold following heat or chemical denaturation. In addition, a number of single domain antibodies have been found to possess high melting points which provide an even greater degree of stability; one of these, llama-derived A3, is a binder of Staphylococcal enterotoxin B and has a Tm of 83.5 °C. In this work, we utilized random mutagenesis and stringent selection in an effort to obtain variants of A3 with even higher melting points. This effort resulted in the selection of a double mutant, A3-T28I-S72I, which has a melting point of 90.0 °C and near wild-type affinity for the target antigen. We further characterized the mutations individually to determine that while both contributed to the thermal stabilization, the T28I mutation accounted for ∼ 4.1 °C of the 6.5 °C increase. This work demonstrates that by the addition of relatively subtle changes it is possible to further improve the melting temperature of single domain antibodies that are already remarkably stable.

Abstract: Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.

Abstract: d-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of l- and d-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host–guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the d-amino acids, including d-allo-isoleucine and d-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate l-amino acids verifies that the intrinsic backbone conformational propensities of the d-amino acids are the inverse of their cognate l-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties.

Abstract: The application of deep sequencing to in vitro display technologies has been invaluable for the straightforward analysis of enriched clones. After sequencing in vitro selected populations, clones are binned into identical or similar groups and ordered by abundance, allowing identification of those that are most enriched. However, the greatest strength of deep sequencing is also its greatest weakness: clones are easily identified by their DNA sequences, but are not physically available for testing without a laborious multistep process involving several rounds of polymerization chain reaction (PCR), assembly and cloning. Here, using the isolation of antibody genes from a phage and yeast display selection as an example, we show the power of a rapid and simple inverse PCR-based method to easily isolate clones identified by deep sequencing. Once primers have been received, clone isolation can be carried out in a single day, rather than two days. Furthermore the reduced number of PCRs required will reduce PCR mutations correspondingly. We have observed a 100% success rate in amplifying clones with an abundance as low as 0.5% in a polyclonal population. This approach allows us to obtain full-length clones even when an incomplete sequence is available, and greatly simplifies the subcloning process. Moreover, rarer, but functional clones missed by traditional screening can be easily isolated using this method, and the approach can be extended to any selected library (scFv, cDNA, libraries based on scaffold proteins) where a unique sequence signature for the desired clones of interest is available.

Abstract: We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both murine and human immunoreactive properties that can be used for in vivo application in murine pre-clinical cancer models.

Abstract: Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose norovirus infections are needed. The goal of this study was to identify antibodies that have broad reactivity of binding to various genogroups of norovirus. A human scFv phage display library was used to identify two antibodies, HJT-R3-A9 and HJT-R3-F7, which bind to both genogroups I and II norovirus virus-like particles (VLPs). Mapping experiments indicated that the HJT-R3-A9 clone binds to the S-domain while the HJT-R3-F7 clone binds the P-domain of the VP1 capsid protein. In addition, a family of scFv antibodies was identified by elution of phage libraries from the GII.4 VLP target using a carbohydrate that serves as an attachment factor for norovirus on human cells. These antibodies were also found to recognize both GI and GII VLPs in enzyme-linked immunosorbent assay (ELISA) experiments. The HJT-R3-A9, HJT-R3-F7 and scFv antibodies identified with carbohydrate elution were shown to detect antigen from a clinical sample known to contain GII.4 norovirus but not a negative control sample. Finally, phages displaying the HJT-R3-A9 scFv can be used directly to detect both GI.1 and GII.4 norovirus from stool samples, which has the potential to simplify and reduce the cost of diagnostics based on antibody-based ELISA methods.

Abstract: Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily cell adhesion molecule that is aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast cancer, colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This wide spectrum of human malignancies makes ALCAM a prospective pan-cancer immunoPET target to aid in detection and diagnosis in multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for 64Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) immunoPET imaging of a fully human ALCAM cys-diabody (cDb) with a reduced linker length that retains its bivalent binding ability. ALCAM constructs with linker lengths of eight, five and three amino acids were produced to make true non-covalent site-specifically modified cDbs. Characterization by gel electrophoresis, size exclusion chromatography, flow cytometry and mass spectrometry of the various constructs was performed. To demonstrate the increased utility of targeting multiple malignancies expressing ALCAM, we compare the targeting of the site-specific versus non-site-specific conjugated cDbs to the human colorectal cancer xenograft LS174T. Interestingly, the conjugation strategy not only affects tumor targeting but also hepatic and renal uptake/clearance.

Abstract: As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.0. Two mutants showing higher catalytic activity at elevated pH in relation to the wild-type XynA were selected, and pooled with a further 5 XynA variants selected by screening thermostable XynA obtained from a previous directed evolution study for activity at alkaline pH. This pool of variants was used as a template for a further round of error-prone polymerase chain reaction and DNase fragment shuffling, with screening at pH 12.0 at 55°C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80°C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80°C, a 3-fold increase in the specific activity compared with the wild-type enzyme at pH 8.0, and a 50% loss of activity (t50) of 60 min at 80°C (wild type <2 min). This directed evolution strategy therefore allows the concomitant adaption of increased thermostability and alkali tolerance of an endo-xylanase.

Abstract: Protein structures available from the PDB contain for each atom the coordinates, the occupancy and the B-factor that indicates the mobility of the atom. The values that should represent B-factors can relate to atomic motions in different ways. We present here a databank in which all B-factors have been converted to the one, homogeneous representation that is most useful for protein engineering applications. The Databank of PDB files with consistent B-factors (BDB) is freely available through http://www.cmbi.umcn.nl/bdb/.

Abstract: Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

Abstract: It is hypothesized that the use of targeted drug delivery systems can significantly improve the therapeutic index of small molecule chemotherapies by enhancing accumulation of the drugs at the site of disease. Phage display offers a high-throughput approach for selection of the targeting ligands. We have successfully isolated phage fusion proteins selective and specific for PANC-1 pancreatic cancer cells. Doxorubicin liposomes (Lipodox) modified with tumor-specific phage fusion proteins enhanced doxorubicin uptake specifically in PANC-1 cells as compared with unmodified Lipodox and also compared with normal breast epithelial cells. Phage protein-targeted Lipodox substantially increased the concentration of doxorubicin in the nuclei of PANC-1 cells in spite of P-glycoprotein-mediated drug efflux. The in vitro cytotoxic activity obtained with pancreatic cell-targeted Lipodox was greater than that of unmodified Lipodox. We present a novel and straightforward method for preparing pancreatic tumor-targeted nanomedicines by anchoring pancreatic cancer-specific phage proteins within the liposome bilayer.

Abstract: To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Abstract: Loops or unordered regions of a protein are structurally dynamic and are strongly implicated in activity, stability and proteolytic susceptibility of proteins. Diminished activity of proteins at lower temperatures is considered to be due to compromised dynamics of the protein at lower temperatures. To evolve an active mesophilic lipase (Bacillus subtilis) at low temperatures, we subjected all the loop residues (n = 88) to site saturation mutagenesis (SSM). Based on a three-level screening protocol, we identified 14 substitutions, among 16,000 mutant population, which contributed to a substantial increase in activity at 5 °C. Based on the preliminary activity of recombinants at several temperatures, 5 substitutions among the 14 were found to be beneficial. A recombinant of these five mutations, named as 5CR, exhibited 7-fold higher catalytic efficiency than wild-type (WT) lipase at 10 °C. All the mutants, individually and in a recombinant (5CR), were characterized by substrate-binding parameters, melting temperatures and secondary structure. 5CR was similar to WT in substrate preferences and showed a significant improvement in activity at both lower and higher temperatures compared with the WT. To establish the contribution of mutations on the dynamics of the protein, we performed 100-ns molecular dynamics (MD) simulations on the WT and mutant lipase at 10 and 37 °C. The root mean square fluctuations (RMSFs) indeed showed that the mutations enhance the protein dynamics locally in the loop region having a catalytic residue, which may help in improved activities at lower temperatures.

Abstract: The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in the cytoplasm of living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, for rapid isolation of intracellular antibodies (intrabodies) in the single-chain Fv format that possess superior traits simply by demanding bacterial growth on high concentrations of antibiotic. Following just a single round of survival-based enrichment using FLI-TRAP, variants of an intrabody against the dimerization domain of the yeast Gcn4p transcription factor were isolated having significantly greater intracellular stability that translated to yield enhancements of >10-fold. Likewise, an intrabody specific for the non-amyloid component region of α-synuclein was isolated that has ~8-fold improved antigen-binding affinity. Collectively, our results illustrate the potential of the FLI-TRAP method for intracellular stabilization and affinity maturation of intrabodies, all without the need for purification or immobilization of the antigen.

Abstract: Non-antibody scaffolds are increasingly used to generate novel binding proteins for both research and therapeutic applications. Our group has developed the tenth fibronectin type III domain of human tenascin-C (TNfn3) as one such scaffold. As a scaffold, TNfn3 must tolerate extensive mutation to introduce novel binding sites. However, TNfn3's marginal stability (T(m) ∼ 59°C, ΔG(unfolding) = 5.7 kcal/mol) stands as a potential obstacle to this process. To address this issue, we sought to engineer highly stable TNfn3 variants. We used two parallel strategies. Using insights gained from structural analysis of other FN3 family members, we (1) rationally designed stabilizing point mutations or (2) introduced novel stabilizing disulfide bonds. Both strategies yielded highly stable TNfn3 variants with T(m) values as high as 83°C and ΔG(unfolding) values as high as 9.4 kcal/mol. Notably, only three or four mutations were required to achieve this level of stability with either approach. These results validate our rational design strategies and illustrate that substantial stability increases can be achieved with minimal mutation. One TNfn3 variant reported here has now been successfully used as a scaffold to develop two promising therapeutic molecules. We anticipate that other variants described will exhibit similar utility.

Abstract: There is an increasing biotechnological interest in 'arming' therapeutic antibodies with bioactive payloads. Many antibody-cytokine fusion proteins (immunocytokines) have been described and some of these biopharmaceuticals have progressed to clinical studies. Here, we describe for the first time the expression and in vivo characterization of immunocytokines based on murine IL1β and IL6. These potent pro-inflammatory cytokines were fused at the N-terminus or at the C-terminus of the monoclonal antibodies F8 (specific to the alternatively-spliced extra-domain A domain of fibronectin, a marker of tumor angiogenesis). All immunocytokines retained the binding properties of the parental antibody and were homogenous, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, except for the N-terminal fusion of IL1β which revealed the presence of glycosylated species. When analyzed by quantitative biodistribution analysis using radioiodinated protein preparations, F8 fusions with IL6 revealed a preferential accumulation at the tumor site for both cytokine orientations, whereas IL1β fusions exhibited lower tumor to organ ratios and a slower blood clearance profile. The fusion proteins with the cytokine payload at the C-terminus were studied in therapy experiments in immunocompetent mice bearing F9 tumors. Immunocytokines based on IL1β resulted in 10% body weight loss at a 5-µg dose, whereas IL6-based products caused a 5% body weight loss at a 225-µg dose. Both F8-IL1β and F8-IL6 exhibited a <50% inhibition of tumor growth rate, which was substantially lower than the one previously reported for F8-TNF, a closely related pro-inflammatory immunocytokine. This study indicates that IL6 can be efficiently delivered to the tumor neo-vasculature by fusion with the F8 antibody. While F8-IL6 was not as potent as other F8-based immunocytokines that exhibit similar biodistribution profiles, the fusion protein sheds light on the different roles of pro-inflammatory cytokines in boosting immunity against the tumor.

Abstract: Basic membrane protein A (BmpA) is a potential carrier protein for surface display of the IgG-binding domain on Lactococcus lactis. We have shown that it can increase the adhesion of bacteria to the intestinal cell model by 1.3-fold and have improved BmpA-based surface display by engineering the BmpA molecule. The bulk of the BmpA molecule was shown to be important in surface display; however, limited shortening (variant Bmp1) resulted in a large increase in the surface display ability. The closeness of the N- and the C-terminals in the Bmp1 model and the inefficiency of the spacer suggest that the distance of the passenger from the membrane is not of prime importance in surface display.

Abstract: The inhibition of ErbB2 by the use of human antibodies can be a valuable strategy for the treatment of breast and gastric cancer. Trastuzumab, a humanized anti-ErbB2 antibody in clinical use, is effective but can engender resistance as well as cardiotoxicity. ImmunoRNases, made up of a human anti-ErbB2 scFv and human pancreatic ribonucleases (HP-RNases), have been engineered to overcome the limits of other immunotoxins, such as immunogenicity and nonspecific toxicity. Here, we report that a novel anti-ErbB2 immunoRNase, called Erb-HPDDADD-RNase, obtained by fusing Erbicin, a human ErbB2-directed scFv, with an HP-RNase variant that resists the cytosolic inhibitor protein, binds with high affinity to a panel of ErbB2-positive gastric tumor cells and inhibits their growth more than does the parental immunoRNase, which is not resistant to the inhibitor. Moreover, Erb-HP-DDADD-RNase is endowed with antiproliferative activity for trastuzumab-resistant cancer cells both in vitro and in vivo that is more potent than that of the parental immunoRNase. Importantly, Erb-HP-DDADD-RNase does not show cardiotoxic effects in vitro on human cardiomyocytes and does not impair cardiac function in a mouse model. Thus, Erb-HP-DDADD-RNase could fulfil the therapeutic need of cancer patients ineligible for trastuzumab treatment due to primary or acquired trastuzumab resistance or to cardiac dysfunction.

Abstract: Artificially transforming a scaffold protein into binders often consists of introducing diversity into its natural binding region by directed mutagenesis. We have previously developed the archaeal extremophilic Sac7d protein as a scaffold to derive affinity reagents (Affitins) by randomization of only a flat surface, or a flat surface and two short loops with natural lengths. Short loops are believed to contribute to stability of extremophilic proteins, and loop extension has been reported detrimental for the thermal and chemical stabilities of mesophilic proteins. In this work, we wanted to evaluate the possibility of designing target-binding proteins based on Sac7d by using a complementary determining region (CDR). To this aim, we inserted into three different loops a 10 residues CDR from the cAb-Lys3 anti-lysozyme camel antibody. The chimeras obtained were as stable as wild-type (WT) Sac7d at extreme pH and their structural integrity was supported. Chimeras were thermally stable, but with T(m)s from 60.9 to 66.3°C (cf. 91°C for Sac7d) which shows that loop extension is detrimental for thermal stability of Sac7d. The loop 3 enabled anti-lysozyme activity. These results pave the way for the use of CDR(s) from antibodies and/or extended randomized loop(s) to increase the potential of binding of Affitins.

Abstract: CCL17 is a homeostatic chemokine associated with several human inflammatory pathologies. This makes CCL17 a potential point of intervention in inflammatory diseases. Using a Fab-pIX phage display system we were able to select antibodies that specifically bind to CCL17 and neutralize CCL17-mediated signaling. Many of the selected antibodies belong to the VH1-69 germline gene family. The VH1-69 germline gene is represented at a high frequency in the human antibody repertoire and is seen in the early immune response to a variety of pathogens. The heavy chain CDR2 of this germline gene is notably hydrophobic and can insert into hydrophobic pockets of antigens, providing much of the binding energy for these antibodies. Affinity maturation of our primary binders by light chain mutagenesis produced antibodies with sub-nanomolar affinities, with affinity improvements up to 100-fold. These were screened for non-specific protein‐protein interactions as a filter for solubility. All of our high affinity antibodies were found to have high levels of non-specific protein‐protein interactions. We speculated that this was due to the hydrophobicity within the germline heavy chain CDR1 and CDR2. To ameliorate this problem, we generated a phage display library for one of the clones, where the surface-exposed residues within H-CDR1 and H-CDR2 were randomized. High stringency panning of this library against human CCL17 resulted in further affinity improvement, along with reduction in protein‐protein interaction in some new variants. In addition, we improved the cross-reactivity to cynomolgus CCL17. We demonstrate that affinity maturation through targeted libraries in the VH1-69 germline gene can improve both affinity and biophysical characteristics of antibodies derived from this gene scaffold.

Abstract: Two LipL32-specific mouse monoclonal antibodies (mAbLPF1 and mAbLPF2) which neutralized Leptospira-mediated hemolysis in vitro and rescued hamsters from lethal Leptospira infection were produced In this communication, locations and characteristics of the protective epitopes of the mAbs were studied by using a truncated LipL32 recombinant protein based-immunoassay and phage consensus mimotope identification and multiple alignments The mAbLPF1 epitope consisted of P243, L244, I245, H246, L252 and Q253 on the LipL32 protein; it is mapped on the surface-exposed region of non-continuous β13-turn and C-terminal amphipathic α6 helix with hydrophobic patch, contributing to phospholipid/host cell adhesion and membrane insertion on one side, and hydrophilic, acidic and basic amino acid residues on another side The epitope peptide of the mAbLPF2 is linear 122PEEKSMPHW130 and located on surface-exposed α1 and α2 between β7 and β8 that bound to several host constituents Both epitopes are highly conserved among the pathogenic and intermediately pathogenic Leptospira spp and are absent from the LipL32 superfamily proteins of other microorganisms This study not only enlightens the molecular mechanisms of the therapeutic mAbLPF1 and mAbLPF2, but also elaborates the potential of the two LipL32 regions as diagnostic and vaccine targets for leptospirosis

Abstract: SurA is a major periplasmic molecular chaperone in Escherichia coli and has been shown to assist the biogenesis of several outer membrane proteins. The C-terminal fragment of SurA folds into a short β-strand, which forms a small three-stranded anti-parallel β-sheet module with the N-terminal β-hairpin. We found that the length of the C-terminal fragment, rather than its exact amino acid composition, had a big impact on SurA function. To investigate the determinant factor of the C-terminal sequence, we created a library of SurA constructs randomized in the last 10 residues. We screened the library and randomly analyzed 19 constructs that displayed SurA activity. The C-termini of these constructs shared little sequence similarity, except that β-strand-forming residues were preferentially enriched. Three SurA constructs were expressed and purified for structural characterization. Circular dichroism and fluorescence spectroscopy analyses revealed that their structures were similar to the structure of the wild-type SurA. Our results suggest that for scaffolding purpose proteins may tolerate various sequences provided certain general requirements such as hydrophobicity and secondary structure propensity are satisfied. Furthermore, the sequence tolerance of SurA at the C-terminus indicates that this area is not likely to be involved in substrate binding.

Abstract: Intrabodies offer attractive options for manipulating the protein misfolding that triggers neurodegenerative diseases. In Huntington's disease, where the expanded polyglutamine tract in the extreme N-terminal region of huntingtin exon1 misfolds, two lead intrabodies have been selected against an adjacent peptide, using slightly different approaches. Both are effective at preventing aggregation of a reporter fragment in transient co-transfection assays. However, after intracranial delivery to mutant mouse brains, VL12.3, which is mainly localized to the nucleus, appears to accelerate the mutant phenotype, while C4 scFv, which is largely cytoplasmic, shows partial phenotypic correction. This comparison highlights parameters that could inform intrabody therapeutics for multiple proteostatic diseases.

Abstract: Due to its frequent overexpression in a variety of solid tumors the epidermal growth factor receptor (EGFR) is a well-established target for therapeutic interventions in epithelial cancers. In order to target EGFR in head and neck cancer, we have generated a ribonuclease (RNase) fusion protein comprising a humanized anti-EGFR antibody single-chain Fv fragment (scFv) and Ranpirnase, an RNase from Rana pipiens. Fusion of Ranpirnase to the N-terminus of the scFv via a flexible glycine-serine linker (G4S)3 resulted in very poor cytotoxicity of the fusion protein. As endosomal accumulation and lysosomal degradation have been reported to diminish the antitumor efficacy of ribonuclease or toxin-based immunoagents, we explored a fusion peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. This peptide was introduced as a linker between Ranpirnase and the scFv moiety. The modified immunoRNase exhibited exceptionally high cytotoxicity toward EGFR-expressing head and neck cell lines without affecting specificity. These results indicate that endosomal entrapment needs to be considered for Ranpirnase-based immunoagents and might be overcome by the use of tailored transduction domains from viral proteins.

Abstract: Cytidine triphosphate (CTP) synthetase (CTPS) (EC number 6.3.4.2) is a key enzyme involved in de novo synthesis of CTP. It catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. In this study, a novel CTPS from Corynebacterium glutamicum ATCC 13032 (CgCTPS) was cloned, expressed and characterized. A series of mutagenesis in its N-terminal ammonia ligase (ALase) domain was performed in order to reduce CTP product inhibition. All single mutation variants (D160E, E162A, E168K) lowered product inhibition by lowering the enzyme's binding affinity for CTP. The homology model of CgCTPS showed that D160E mutant caused steric hindrance for the pyrimidine ring of CTP stacking, E162A disrupted the hydrogen bond between CTP ribose and side chain and D168K caused minor localized structure perturbations of CTP binding pocket. The triple mutant of CTPS (D160E-E162A-E168K) with halved Km, doubled Vmax and the 23.5-fold increased IC50 for CTP shows a potential for use in industrial-scale CTP production by its better performance in enzyme kinetics and product inhibition.