Prediction of antigen-responding VHH antibodies by tracking the evolution of antibody along the time course of immunization.

Antibody maturation is the central function of the adaptive immune response. This process is driven by the repetitive selection of mutations that increase the affinity toward antigens. We hypothesized that a precise observation of this process by high-throughput sequencing along the time course of immunization will enable us to predict the antibodies reacting to the immunized antigen without any additional in vitro screening. An alpaca was immunized with IgG fragments using multiple antigen injections, and the antibody repertoire development was traced via high-throughput sequencing periodically for months. The sequences were processed into clusters, and the antibodies in the 16 most abundant clusters were generated to determine whether the clusters included antigen-binding antibodies. The sequences of most antigen-responsive clusters resembled those of germline cells in the early stages. These sequences were observed to accumulate significant mutations and also showed a continuous sequence turnover throughout the experimental period. The foregoing characteristics gave us >80% successful prediction of clusters composed of antigen-responding VHHs against IgG fragment. Furthermore, when the prediction method was applied to the data from other alpaca immunized with epidermal growth factor receptor, the success rate exceeded 80% as well, confirming the general applicability of the prediction method. Superior to previous studies, we identified the immune-responsive but very rare clusters or sequences from the immunized alpaca without any empirical screening data.

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag.

In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

Immune response with long-term memory triggered by amorphous aggregates of misfolded anti-EGFR VHH-7D12 is directed against the native VHH-7D12 as well as the framework of the analogous VHH-9G8.

We previously demonstrated that amorphous aggregates of misfolded VHH-7D12 antibodies (VHH-Mis), a potential anti-EGFR drug, can generate a robust serum IgG response. Here we investigate the immunogenic nature, especially the specificity of the immune response induced by VHH-Mis. To this end, we used two natively folded and 77% identical anti-EGFR VHHs (VHH-7D12 and VHH-9G8) that possess a common framework but distinct complementarity determining regions (CDRs). In 60% of mice immunized with VHH-Mis, the anti-VHH-7D12 IgG titer was stronger than the anti-VHH-9G8 titer (Group-1). In the remaining mice (40%; Group-2), the anti-VHH-7D12 and anti-VHH-9G8 titer were almost identical. We rationalized these results by hypothesizing that mice in Group-1 produced IgG mostly against the VHH-7D12's CDRs, whereas in Group-2 mice, they targeted the VHH's framework. The IgG specificity against VHH-7D12 and VHH-9G8 was essentially unchanged over 17 weeks in both groups. Further, in all mice (Group-1&2) re-immunized with native VHH-7D12, the IgG titer against VHH-7D12 increased sharply but not against VHH-9G8. On the other hand, none of the three Group-1 mice re-immunized with native VHH-9G8 showed immunogenicity against VHH-7D12 nor VHH-9G8. Whereas, in Group-2 mice (three/three) re-immunized with VHH-9G8, the IgG titers against both VHHs increased but slowly. Flow-cytometric studies showed that VHH-Mis immunized mice generated a higher number of effector and central memory T-cells. Overall, these observations indicate that amorphous aggregates made of a misfolded VHH can induce serum IgG against its natively folded self and analogous VHHs having a similar framework but distinct CDRs. Furthermore, a robust long-term immune response with memory was established against its natively folded self but with a nil-to-moderate immune response against natively folded VHH analogs.

Breakdown of supersaturation barrier links protein folding to amyloid formation.

The thermodynamic hypothesis of protein folding, known as the "Anfinsen's dogma" states that the native structure of a protein represents a free energy minimum determined by the amino acid sequence. However, inconsistent with the Anfinsen's dogma, globular proteins can misfold to form amyloid fibrils, which are ordered aggregates associated with diseases such as Alzheimer's and Parkinson's diseases. Here, we present a general concept for the link between folding and misfolding. We tested the accessibility of the amyloid state for various proteins upon heating and agitation. Many of them showed Anfinsen-like reversible unfolding upon heating, but formed amyloid fibrils upon agitation at high temperatures. We show that folding and amyloid formation are separated by the supersaturation barrier of a protein. Its breakdown is required to shift the protein to the amyloid pathway. Thus, the breakdown of supersaturation links the Anfinsen's intramolecular folding universe and the intermolecular misfolding universe.

The immunogenicity of an anti-EGFR single domain antibody (VHH) is enhanced by misfolded amorphous aggregation but not by heat-induced aggregation.

Amorphous aggregates of therapeutic proteins can provoke an unwanted immune response (anti-drug antibodies; ADAs), but counter-examples have led to some controversy. Amorphous aggregates can possess unique biophysical and biochemical attributes depending on both the way they are generated and the protein's biophysical/biochemical properties. Here, we examine the immunogenicity of an anti-EGFR single domain antibody (VHH) in four types of amorphous aggregates: two heat-aggregated VHH incubated at 65 °C (VHH-65) and 95 °C (VHH-95), a misfolded VHH isolated from the insoluble fraction of the E. coli lysate (VHH-Ins), and a low solubility misfolded VHH produced by miss-shuffling the SS bonds of the native VHH (VHH-Mis). Biophysical and biochemical measurements indicated that VHH was indeed natively folded, monomeric, and ß-sheeted; that VHH-65 was partially unfolded and formed aggregates with a Z-average (Zave) of 771 nm; whereas VHH-95 was unfolded and formed aggregates of 1722 nm; and that both VHH-Ins and VHH-Mis were misfolded with non-native intermolecular SS bonds and formed aggregates with a Zave of 1846 nm and 1951 nm, respectively. The IgG level generated in Jcl:ICR mice determined by ELISA showed that the native VHH was barely immunogenic, VHH-95 was not immunogenic, while VHH-65 was mildly immunogenic. By contrast, the misfolded aggregates, VHH-Ins and VHH-Mis, having a Zave and an aggregation propensity similar to that of VHH-95, were highly immunogenic. These findings indicate the critical role of the biochemical and biophysical attributes of the amorphous aggregates in generating an immune response against a protein, rather than just their sizes.

PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody.

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody-drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single-domain VHH (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product-single-stranded DNA conjugated at the carboxyl terminus of VHH-retained its affinity for EGFR. To investigate whether this VHH-single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100-500 bp DNA. We confirmed the amplification of the VHH-double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.

Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility.

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20 °C, ∼85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15 mg which was ∼3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37 °C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.

Anti-survivin single-domain antibodies derived from an artificial library including three synthetic random regions by in vitro selection using cDNA display.

Single-domain antibodies (variable domain of the heavy chain of a heavy chain antibody; VHH) are promising reagents for therapeutics and diagnostics because of their stability, cost-effective production and material workability as a small antibody. Currently, general acquisition of a VHH using immunization of camelids is inconvenient from the standpoint of animal protection, cost and the process is time-consuming. Thus, a straightforward and efficient method for screening VHHs against a target molecule is required. In this study, we examined whether in vitro selection of a VHH against a target protein could be performed by a cDNA display method with an artificial VHH library that had the three complementarity-determining regions (CDRs) randomized by chemical synthesis. The results revealed that a particular VHH against survivin, which is a member of the inhibitor of apoptosis family, was selected with affinity in the range of 10-7 to 10-8 M. The in vitro selection of a VHH using cDNA display with an artificial synthesized library without animal immunization was shown to be effective for rapid and inexpensive screening of VHHs against a target protein.

Heat-Induced Aggregation of Hen Ovalbumin Suggests a Key Factor Responsible for Serpin Polymerization.

Although ovalbumin (OVA), a main component of hen egg white and a non-inhibitory serpin superfamily protein, has been reported to form fibrillar aggregates, its relationship with amyloid fibrils associated with various degenerative diseases is unclear. We studied the heat-induced aggregation of intact OVA using an amyloid-specific thioflavin T assay with a fluorometer or direct imaging with a light-emitting diode lamp and several physicochemical approaches, and the results confirmed that intact OVA forms aggregates with a small part of amyloid cores and dominantly amorphous aggregates. We isolated the amyloidogenic core peptide by proteolysis with trypsin. The isolated 23-residue peptide, pOVA, with marked amyloidogenicity, corresponded to one (ß-strand 3A) of the key regions involved in serpin latency transition and domain-swap polymerization leading to serpinopathies. Although the strong amyloidogenicity of pOVA was suppressed in a mixture of tryptic digests, it was observed under acidic conditions in the presence of various salts, with which pOVA has a positive charge. Cytotoxicity measurements suggested that, although heat-treated OVA aggregates exhibited the strongest toxicity, it was attributed to a general property of amorphous aggregates rather than amyloid toxicity. Predictions indicated that the high amyloidogenicity of the ß-strand 3A region is common to various serpins. This suggests that the high amyloidogenicity of ß-strand 3A that is important for serpin latency transition and domain-swap polymerization is retained in OVA and constitutes ß-spine amyloid cores upon heat aggregation.

Rapid Enzyme-linked Immunosorbent Assays for Diagnosis of Diabetes in a Compact Disc-shaped Microfluidic Device.

We have developed a compact disc (CD)-shaped microfluidic device for multiple, rapid enzyme-linked immunosorbent assays (ELISA). The device has a versatile design that can be adapted for the detection of various proteins by selecting the push-in-type reaction parts and appropriate reagents for each target. In this paper, we report the rapid quantification of insulin, adiponectin, and leptin, which can be used for the early diagnosis of diabetes, in human serum in only 16 min with our device.

Heat denaturation of the antibody, a multi-domain protein.

The antibody is one of the most well-studied multi-domain proteins because of its abundance and physiological importance. In this article, we describe the effect of the complex, multi-domain structure of the antibody on its denaturation by heat. Natural antibodies are composed of 6 to 70 immunoglobulin fold domains, and are irreversibly denatured at high temperatures. Although the separated single immunoglobulin fold domain can be refolded after heat denaturation, denaturation of pairs of such domains is irreversible. Each antibody subclass exhibits a distinct heat tolerance, and IgE is especially known to be heat-labile. IgE starts unfolding at a lower temperature compared to other antibodies, because of the low stability of its CH3 domain. Each immunoglobulin domain starts unfolding at different temperatures. For instance, the CH3 domain of IgG unfolds at a higher temperature than its CH2 domain. Thus, the antibody has a mixture of folded and unfolded structures at a certain temperature. Co-existence of these folded and unfolded domains in a single polypeptide chain may increase the tendency to aggregate which causes the inactivation of the antibody.

Oxidation and interaction of DJ-1 with 20S proteasome in the erythrocytes of early stage Parkinson's disease patients.

Parkinson's disease (PD) is a progressive, age-related, neurodegenerative disorder, and oxidative stress is an important mediator in its pathogenesis. DJ-1, the product of the causative gene of a familial form of PD, plays a significant role in anti-oxidative defence to protect cells from oxidative stress. DJ-1 undergoes preferential oxidation at the cysteine residue at position 106 (Cys-106) under oxidative stress. Here, using specific antibodies against Cys-106-oxidized DJ-1 (oxDJ-1), it was found that the levels of oxDJ-1 in the erythrocytes of unmedicated PD patients (n = 88) were higher than in those of medicated PD patients (n = 62) and healthy control subjects (n = 33). Elevated oxDJ-1 levels were also observed in a non-human primate PD model. Biochemical analysis of oxDJ-1 in erythrocyte lysates showed that oxDJ-1 formed dimer and polymer forms, and that the latter interacts with 20S proteasome. These results clearly indicate a biochemical alteration in the blood of PD patients, which could be utilized as an early diagnosis marker for PD.

Thioflavin T-Silent Denaturation Intermediates Support the Main-Chain-Dominated Architecture of Amyloid Fibrils.

Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of ß2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than ß2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation.

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies.

Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.

Cytotoxicity of CdSe-based quantum dots incorporated in glass nanoparticles evaluated using human keratinocyte HaCaT cells.

CdSe quantum dots (QDs) are potential fluorescent reagents, but leakage of Cd and Se often induces cytotoxicity. Here we prepared CdSe-based QDs with glass to reduce their leakage and examined their cytotoxicity using keratinocyte cells. The cytotoxicity of the QDs with glass was obviously lower than that of the commercial QDs with polymer, suggesting their safety for biological applications.

Singlet-oxygen-derived products from linoleate activate Nrf2 signaling in skin cells.

Linoleates are required for normal mammalian health and development, but they are also prone to oxidation, resulting in biologically active metabolites such as hydroxyoctadecadienoic acids (HODEs). To investigate the biological activity of 9-EZ-HODE, 10-EZ-HODE, 12-ZE-HODE, and 13-ZE-HODE, the metabolites of singlet-oxygen-derived products from linoleates, we assessed adaptive cytoprotection in HaCaT skin cells. Treating HaCaT cells with sublethal concentrations of 10-EZ-HODE and 12-ZE-HODE, which are singlet-oxygen-mediated specific oxidation metabolites of linoleates, but not 9-EZ-HODE and 13-ZE-HODE, caused resistance to hydrogen peroxide-induced oxidative damage. Microarray analysis of HaCaT cells revealed that 10-EZ-HODE and 12-ZE-HODE induced cellular antioxidant genes that are responsive to nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), such as heme oxygenase-1 and glutathione synthesis enzymes. Although 10-EZ-HODE and 12-ZE-HODE did not induce Nrf2 mRNA, treatment with these metabolites increased the intranuclear expression of Nrf2. These results suggest that 10-EZ-HODE and 12-ZE-HODE initiate adaptive responses that reduce the damage caused by oxidative stress.

Ultrasonication-dependent formation and degradation of α-synuclein amyloid fibrils.

Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinson's disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered.

Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications.

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

The effect of oxycholesterols on thermo-induced membrane dynamics.

The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7ß-hydroxycholesterol (7ß) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7ß; (iii) 7ß and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.