A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

Exploiting sequence and stability information for directing nanobody stability engineering

BackgroundVariable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein.MethodsWe analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally.ResultsSome mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated.ConclusionsThe results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-specificity of nanobody architecture.General significanceThis study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Interactions of bovine fibrinogen and thrombin. Early events in the development of clot structure.

Interactions which determine the rate of conversion of fibrinogen into monomer fibrin and the retention of monomer fibrin in a noncompactible form through interaction with residual fibrinogen (solution stabilization) were examined through the kinetics of formation of equilibrium compactible network at pH 7 and ionic strength 0.15. For studies of conversion, reactions with thrombin were at 29 or 2 °C, hirudin was added at successive times to inhibit thrombin, and compactible network was equilibrated at 2 °C, where solution stabilization is negligible. A substrate dependency of initial rate is interpreted on the basis of inactive complex formation between thrombin and both fibrinogen and monomer fibrin. At 29 or 2 °C specific rate constants are 32 or 2.9 × 10⁶ liter/mol, and association constants for inactive complex formation are 5.2 or 2.0 × 10⁵ liter/mol. The second peptide-A is removed from fibrinogen ~ 40-fold as rapidly as the first.With equilibration at 29 °C, compactible network does not appear until the solution stabilization ratio of residual fibrinogen/monomer fibrin is four. Thereafter, increasing amounts of compactible network appear. However, the stabilization ratio progressively decreases to approximately two, a situation which indicates the complexity of the stabilization mechanism.The thrombin-hirudin association constant is estimated to be 4.9 or 17 × 10¹¹ liter/mol at 29 or 2 °C.     

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

ABSTRACT

Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (V_L) and heavy (V_H) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, we are able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, we could isolate a diverse panel of >40 unique antigen-binding variants. Additionally, we successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.     

The effect of stimulus number on the stability of responses for an extensive heat pain test

Background.?The use of relatively lower stimulus presentation numbers in quantitative sensory testing may influence the computation accuracy of participants’ discriminability. The minimum trial number for obtaining a stabilized participant discrimination ability was determined.

Materials and methods.?Twelve participants’ ability to discriminate between noxious heat stimuli pairs (45°C/46°C, 46°C/47°C, and 47°C/48°C) was assessed using a six-category confidence rating scale. Heat stimuli were administered to the forearm. Two conditions with presentation numbers of 17 trials per stimulus (representing the median number of trials in previous studies) and 40 trials per stimulus (used in a previous study with a similar protocol) were used.

Results and discussion.?Participants’ discriminability stabilized at approximately the 20th trial based on the lowest frequency of indeterminate and non-model conforming results under both conditions. A simple linear regression model showed a statistically significant positive relationship between discriminability for the two conditions (slope?=?0.65, p?<?0.001; constant?=?0.33, p?=?0.02; r^2?=?0.51). As a rule of thumb, approximately 20 trials per stimulus intensity could be used to obtain a stabilized discriminability outcome.     

Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization of the receptor

The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and ¹²⁵l-labeled anti-transferrin-receptor antibodies. Vesicles containing peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with anti-transferrin-receptor anti-bodies. Vesicle formation does not require the presence of the anti-transferrin-receptor antibodies. Using ¹²⁵l-surface-labeled reticulocytes, it can be shown that the ¹²⁵l-labeled material which is released is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticulocytes tagged with ¹²⁵l-labeled anti-transferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0°C or in phosphate-buffered saline the rate of vesicle release is less than that at 37°C in culture medium. There is selective externalization of the antibody-receptor complex since few other membrane proteins are found in the externalized vesicles. The anti-transferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.     

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

Temperature requirements for the growth and maturation of the gametophytes of Undaria pinnatifida and U. undarioides (Laminariales, Phaeophyceae)

Gametophytes of two Undaria species, U. pinnatifida and U. undarioides (Laminariales, Phaeophyceae), were studied to determine their water temperature requirements in order to understand their different distributions in Mie Prefecture, Japan. The optimal temperature for growth was 20°C for gametophytes of both species, and the upper critical temperature for growth was also the same for both species at 28°C. Therefore, the optimal and critical temperatures for growth of the gametophytes are not the main factors determining distribution. The optimal temperature for maturation of U. pinnatifida was approximately 10–15°C, whereas it was closer to 20–21°C for U. undarioides, a difference between these species of at least 5°C. In autumn and early winter, the seawater temperature at the mouth of Ise Bay, where U. pinnatifida is distributed, ranges from 21.6°C (October) to 12.7°C (December), and off Hamajima, where U. undarioides is found, the range is from 22.7°C (October) to 19.1°C (December). The seawater temperatures from October to December, which is the maturation season for the gametophytes, agreed well with the optimal temperature requirements for maturation of the gametophytes of both species. Thus the difference in the maturation temperature range of the gametophytes is a major factor determining distribution of these Undaria species along the Japanese coast.     

Structural Studies of 2-Thiouridine in RNA

Abstract

A pentamer RNA sequence, Gs²UUUC, and a s²U containing 14-mer RNA tetraloop hairpin were synthesized and characterized by NMR and by UV melting studies. These oligonucleotides were used as models to understand the effect of 2-thiouridine substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through sU-34 modification. The magnitude of the effect of sU in our model system is comparable to the 20° C stabilization reported for 2-thiolation in a codonanticodon model system composed of two tRNAs with complementary anticodon sequences.     

E-rosette formation at 37 °C: Analysis with monoclonal OKT antibodies

When cultured in the presence of PHA, a proportion of human peripheral blood mononuclear cells acquires the capacity to form E rosettes with sheep erythrocytes that are resistant to incubation at 37 °C. The nature of this 37 °C stable E-rosette formation was investigated using a panel of monoclonal OKT antibodies directed to human T-lymphocyte surface antigens. OKT11A antibody, at a concentration of 0.2–0.4 μg/ml, markedly blocked 37 °C E rosetting. OKT1, OKT3, OKT4, OKT6, and OKT8 antibodies, when tested at 10 μg/ml, show no such inhibiting activity. Quantitative studies with ¹²⁵I-labeled OKT11A indicated that the antibody interacted strongly with both 37 °C E-rosetting and nonrosetting cells, the association constant being 1.6–2.0 × 10⁹M^?1. However, on the average, a threefold higher concentration of OKT11A receptor sites was found on 37 °C E-rosette-forming cells (14.8 × 10⁴ sites/cell) than on nonrosetting cells (4.8 × 10⁴ sites/cell). Our data suggest that 37 °C E-rosette formation is governed by a lymphocyte surface determinant recognized by OKT11A antibody. “Overexpression” of OKT11A antigenic sites on a proportion of PHA-stimulated lymphocytes may explain their capacity to form 37 °C stable E-rosettes.     

Stabilization of cyclohexanone monooxygenase by computational and experimental library design

Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature-sensitive enzymes catalyzing the Baeyer–Villiger reaction, which display excellent stereo- and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI-31, a substrate-promiscuous enzyme that efficiently catalyzes the production of the nylon-6 precursor ε-caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein-stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half-life at 30°C. In contrast to the wild-type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications.     

Characterization of CaMKIIα holoenzyme stability

Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long‐term memory formation and other Ca²⁺‐dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X‐ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T_m = 36°C), which is slightly stabilized by ATP/MgCl₂ binding (apparent T_m = 40°C) and significantly stabilized by regulatory segment binding (apparent T_m = 60°C). We crystallized the kinase domain of CaMKII bound to p‐coumaric acid in the active site. This structure reveals solvent‐exposed hydrophobic residues in the substrate‐binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T_m ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.     

抗体片段在铂纳米粒子表面的构象重构

目的 最近在金纳米粒子（AuNPs）表面重构抗体片段的天然构象和功能的研究表明分子构象工程的可行性。本质上,分子构象工程就是要像蛋白质折叠一样,通过精确控制柔性非功能分子的构象使其产生新功能。本文在铂纳米粒子（PtNPs）表面重构抗体互补决定簇区（CDR）片段的天然构象和功能,旨在探索分子构象工程的普适性及揭示蛋白质结构-功能机制。方法 本文将抗溶菌酶抗体（cAB-lys3）中的CDR3片段（在单独存在时没有稳定构象和功能）通过两个Pt-S键偶联到PtNPs表面。CDR片段的天然构象和功能的恢复通过它对溶菌酶活性的抑制来表征。结果 通过多肽密度优化和表面聚乙二醇修饰,制得基于PtNPs的抗溶菌酶人工抗体（简称铂抗体）。溶菌酶活性测试结果表明,铂抗体可以特异性结合溶菌酶并显著抑制其活性。结论 本文第一次在PtNPs表面重构了蛋白质片段的天然构象并恢复了其功能,证明分子构象工程可作为一种通用方法制备基于纳米粒子的人工蛋白质。     

Elevated temperature increases the accumulation of microbial necromass nitrogen in soil via increasing microbial turnover

Microbial‐derived nitrogen (N) is now recognized as an important source of soil organic N. However, the mechanisms that govern the production of microbial necromass N, its turnover, and stabilization in soil remain poorly understood. To assess the effects of elevated temperature on bacterial and fungal necromass N production, turnover, and stabilization, we incubated ¹⁵N‐labeled bacterial and fungal necromass under optimum moisture conditions at 10°C, 15°C, and 25°C. We developed a new ¹⁵N tracing model to calculate the production and mineralization rates of necromass N. Our results showed that bacterial and fungal necromass N had similar mineralization rates, despite their contrasting chemistry. Most bacterial and fungal necromass ¹⁵N was recovered in the mineral‐associated organic matter fraction through microbial anabolism, suggesting that mineral association plays an important role in stabilizing necromass N in soil, independently of necromass chemistry. Elevated temperature significantly increased the accumulation of necromass N in soil, due to the relatively higher microbial turnover and production of necromass N with increasing temperature than the increases in microbial necromass N mineralization. In conclusion, we found elevated temperature may increase the contribution of microbial necromass N to mineral‐stabilized soil organic N.     

Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.     

Nocturnal parasitism of moth eggs by Trichogramma wasps

Parasitoid wasps of the genus Trichogramma are used worldwide as biological control agents against lepidopteran pests. Trichogramma wasps develop inside eggs of a wide range of host species, most of them moths. They are generally considered as diurnal insects. Here, we investigated whether Trichogramma wasps can also successfully parasitise host eggs at night under controlled laboratory conditions. Eggs of the moth Ephestia kuehniella were offered under dark conditions (scotophase) to females of Trichogramma brassicae and Trichogramma evanescens either from 9:00 PM to 9:00 AM or from 11:00 AM to 5:00 PM at four different temperatures (5°C, 10°C, 15°C and 20°C). Both species are known to parasitise E. kuehniella eggs in the photophase during daytime. The results show that T. brassicae did not parasitise eggs in the scotophase at night and only very few in the artificially induced scotophase during daytime from 10°C to 20°C. In contrast, T. evanescens parasitised more eggs in the dark both at night and artificially induced scotophase during daytime. Parasitism in the scotophase already started at 5°C, with more eggs being parasitised and more offspring being produced at higher temperatures. T. evanescens displayed higher parasitism activity in the induced scotophase during daytime than in the scotophase at night. The present study suggests that Trichogramma are capable of successfully parasitising host eggs at night, even at low temperatures, but that nocturnal activity with respect to parasitism varies between wasp species.     

Geological cycles

Abstract

Deep body temperature (DBT) and heart rate (HR) circadian rhythms were determined by radiotelemetry in 4 mares kept under controlled light and temperature conditions. Ovulations were determined by rectal palpation of their ovaries. Mean DBT values ranged from 35.85 ± .04 to 37.22 ± .02°C The circadian range of oscillation was extremely low, approximately 0.5° C, with time of maximum temperature occurring midway through the dark period. Mean HR values ranged from 36.4 ± 1.7 to 53.0 ±3.6 beats per min. The circadian range of oscillation was also low, less than 15 beats per min with time of maximum HR occurring approximately at the time of lights off. The HR circacadian rhythm peaked before the DBT circadian rhythm by 3 to 8 hrs. Ovulation did not appear to consistently affect DBT and HR circadian rhythms or their phase relationships.     

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.     

Experimental evaluation of single‐domain antibodies predicted by molecular dynamics simulations to have elevated thermal stability

Recently Bekker et al. [Bekker G‐J et al. Protein Sci. 2019;28:429–438.] described a computational strategy of applying molecular‐dynamics simulations to estimate the relative stabilities of single‐domain antibodies, and utilized their method to design changes with the aim of increasing the stability of a single‐domain antibody with a known crystal structure. The structure from which they generated potentially stabilizing mutations is an anti‐cholera toxin single domain antibody selected from a naïve library which has relatively low thermal stability, reflected by a melting point of 48°C. Their work was purely theoretical, so to examine their predictions, we prepared the parental and predicted stabilizing mutant single domain antibodies and examined their thermal stability, ability to refold and affinity. We found that the mutation that improved stability the most (~7°C) was one which changed an amino acid in CDR1 from an asparagine to an aspartic acid. This change unfortunately was also accompanied by a reduction in affinity. Thus, while their modeling did appear to successfully predict stabilizing mutations, introducing mutations in the binding regions is problematic. Of further interest, the mutations selected via their high temperature simulations, did improve refolding, suggesting that they were successful in stabilizing the structure at high temperatures and thereby decrease aggregation. Our result should permit them to reassess and refine their model and may one day lead to a usefulin silico approach to protein stabilization.