Nanobody-Based high-performance immunosorbent for selective beta 2-microglobulin purification from blood.

Removing ß2-microglobulin (ß2M) from blood circulation is considered to be the most effective method to delay the occurrence of dialysis-related amyloidosis (DRA). The ideal extracorporeal ß2M removal system should be cost-effective, highly specific and having a high capacity. However, the traditional technologies based on size exclusion do not have an adequate specificity, and alternative immunosorbents have limited applications due to low capacity and their high cost. Nanobodies (Nbs), the smallest functional recombinant antibody fragments, offer several advantages to overcome these obstacles. In this study, an anti-ß2M Nb with a C-terminal thiol-tag was successfully prepared from E. coli for site-directed and oriented immobilization and usage as capture ligand in a ß2M-selective immunosorbent. The prepared immunosorbent showed a high binding capacity of up to 7 mg ß2M per mL resin, which is 17 times higher than that of previous studies using single-chain variable antibody fragments (scFv). Furthermore, an exceptional high specificity has been demonstrated as other human serum proteins were not adsorbed during dynamic adsorption experiments. About 80% of the original binding capacity of the immunosorbent was restored after four consecutive easy regenerations, whereas 90% of the original capacity was retained after 1-month storage of the resin. Moreover, the mathematical model fitted very well the in vitro perfusion. The results with this pioneering immunosorbent confirm its possible clinical application and is expected to reach the required clinical effect of immunoadsorption therapy. STATEMENT OF SIGNIFICANCE: Dialysis-related amyloidosis (DRA), associated with the accumulation of ß2-microglobulin (ß2M), is a serious complication of end-stage kidney disease. Removing ß2M from blood circulation by extracorporeal blood purification is considered to be the most effective method to delay the occurrence of DRA. However, the existing methods are incapable to eliminate sufficient quantities of ß2M from circulation, either because of lack of specificity, high cost or for low capacity. In this manuscript, we provide a practical and economic immunosorbent based on anti-ß2M nanobody for DRA. The prepared immunosorbent was reusable and storable, and demonstrated high specificity and realized a high binding capacity of up to 7 mg ß2M per mL resin, which is 17 times higher than that of the previous studies.

High-permeability alternatives to current dialyzers performing both high-flux hemodialysis and postdilution online hemodiafiltration.

Most high-flux dialyzers can be used in both hemodialysis (HD) and online hemodiafiltration (OL-HDF). However, some of these dialyzers have higher permeability and should not be prescribed for OL-HDF to avoid high albumin losses. The aim of this study was to compare the safety and efficacy of a currently used dialyzer in HD and OL-HDF with those of several other high permeability dialyzers which should only be used in HD. A prospective, single-center study was carried out in 21 patients. Each patient underwent 5 dialysis sessions with routine dialysis parameters: 2 sessions with Helixone (HD and postdilution OL-HDF) and 1 session each with steam sterilized polyphenylene, polymethylmethacrylate (PMMA), and medium cut-off (MCO) dialyzers in HD treatment. The removal ratios (RR) of urea, creatinine, ß2 -microglobulin, myoglobin, prolactin, α1 -microglobulin, α1 -acid glycoprotein, and albumin were compared intraindividually. A proportional part of the dialysate was collected to quantify the loss of various solutes, including albumin. Urea and creatinine RRs with the Helixone-HDF and MCO dialyzers were higher than with the other 3 dialyzers in HD. The ß2 -microglobulin, myoglobin and prolactin RRs with Helixone-HDF treatment were significantly higher than those obtained with all 4 dialyzers in HD treatment. The ß2 -microglobulin value obtained with the MCO dialyzer was also higher than that obtained with the other 3 dialyzers in HD treatment. The myoglobin RR with MCO was higher than those obtained with Helixone and PMMA in HD treatment. The prolactin RR with Helixone-HD was significantly lower than those obtained in the other 4 study sessions. The α1 -microglobulin and α1 - acid glycoprotein RRs with Helixone-HDF were significantly higher than those obtained with Helixone and PMMA in HD treatment. The albumin loss varied from 0.54 g with Helixone-HD to 3.3 g with polyphenylene. The global removal score values ((UreaRR + ß2 -microglobulinRR + myoglobinRR + prolactinRR + α1 -microglobulinRR + α1 -acid glycoproteinRR - albuminRR )/6) were 43.7% with Helixone-HD, 47.7% with PMMA, 54% with polyphenylene, 54.8% with MCO and 59.6% with Helixone-HDF, with significant differences. In conclusion, this study confirms the superiority of OL-HDF over HD with the high-flux dialyzers that allow both treatments. Although new dialyzers with high permeability can only be used in HD, they are in an intermediate position and some are very close to OL-HDF.

Clinical Benefit of an Adsorptive Technique for Elderly Long-Term Hemodialysis Patients.

BACKGROUND: With the advancement of technology, a dialysis membrane has been developed to achieve the efficient removal of beta-2 microglobulin (ß2MG), which could not be removed with previous hemodialysis (HD) membranes. Recently, there has been an increase in the population of elderly chronic kidney disease (CKD) patients with chronic inflammation and malnutrition. The optimal extracorporeal circulation treatment for elderly CKD patients is not certain. SUMMARY: We have reported the clinical advantages, such as improvements in nutritional, inflammatory, and hemodynamic conditions, of the adsorptive HD membrane for elderly HD patients. We have also reported that the use of ß2MG adsorption columns improved the symptoms of dialysis-related amyloidosis and the number of bone cysts, which could not be improved by the high-flux hemodialyzer. Both the adsorptive HD membrane and ß2MG adsorption columns remove uremic toxins and inflammatory cytokines via adsorption without aggravating the nutritional condition of these patients. Key Messages: We should reconsider the mechanisms of adsorption, in addition to diffusion and convection, in the extracorporeal circulation treatment of elderly HD patients.

Novel polystyrene/antibody nanoparticle-coated capillary for immunoaffinity in-tube solid-phase microextraction.

Antibody-coated polystyrene (PS) nanoparticles (denoted as PS/IgG) were prepared and chemically immobilized for the first time onto a capillary inner wall for immunoaffinity in-tube solid-phase microextraction (SPME) of ß2-microglobin (ß2MG) and cystatin C (Cys-C). Scanning electron microscopy images of the prepared capillary showed that the nanoparticles were evenly coated onto the capillary inner surface, resulting in the undulating surface of the capillary inner wall. The extraction capacity of the nanoparticle-coated capillary was nearly five times higher than that of a monolayer antibody-immobilized capillary. The in-tube SPME recovery of ß2MG (or Cys-C) by the nanoparticle-functionalized capillary was more than 97.8%, whereas that by the monolayer antibody-immobilized tube was 30.5%. In addition, the method quantitation limit obtained by using the nanoparticle-coated capillary was ten times lower than that by the monolayer capillary. Therefore, the capillary coated by PS/IgG nanoparticles is more suitable for immunoaffinity in-tube SPME compared with the commonly used monolayer antibody-immobilized capillary.

Novel antibacterial activity of ß(2)-microglobulin in human amniotic fluid.

An antibacterial protein (about 12 kDa) was isolated from human amniotic fluid through dialysis, ultrafiltration and C18 reversed-phase HPLC steps. Automated Edman degradation showed that the N-terminal sequence of the antibacterial protein was NH(2)-Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-. The N-terminal sequence of the antibacterial protein was found to be identical to that of ß(2)-microglobulin, a component of MHC class I molecules, which are present on all nucleated cells. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) revealed that the molecular mass of the antibacterial protein was 11,631 Da. This antibacterial protein, ß(2)M, possessed potent antibacterial activity against pathogenic bacteria. Specially, antibacterial activity was observed in potassium buffer, and potassium ion was found to be critical for the antibacterial activity. Interestingly, the antibacterial action of ß(2)M was associated with dissipation of the transmembrane potential, but the protein did not cause damage to the membrane that would result in SYTOX green uptake. In addition, stimulation of WISH amniotic epithelial cells with the bacterial endotoxin lipopolysaccharide (LPS) induced dose-dependent upregulation of ß(2)M mRNA expression. These results suggest that ß(2)M contributes to a self-defense response when amniotic cells are exposed to pathogens.

Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and ß2M-sfGFP.

The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and ß2-microglobulin (ß2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and ß2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and ß2M that are suitable for further studies.

Removal of intact ß2-microglobulin at neutral ph by using seed-conjugated polymer beads prepared with ß2-microglobulin-derived peptide (58-67).

Removal of ß2-microglobulin (ß2M) from the blood of patients suffering from kidney dysfunction is crucial to protect those individuals from getting the diseased state of dialysis-related amyloidosis. By harnessing the nucleation-dependent fibrillation process of amyloidogenesis, a ß2M removal strategy has been proposed by preparing seed-conjugated polymer beads and assimilating soluble ß2M to the fibrils on the surface at neutral pH. A novel peptide segment of ß2M ranging from residue 58 to residue 67 (Lys-Asp-Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr), which was capable of being fibrillated at neutral pH was isolated. Charge interaction between the positive N-terminal lysine and the negative C-terminal α-carboxylic group was demonstrated to be critical for the molecular self-assembly leading to the peptide fibril formation by favoring ß-sheet conformation. Because the peptide fibrils were successful to seed intact ß2M at neutral pH, the fibrils were immobilized on polymer beads of HiCore resins, and the resulting seed-conjugated beads were used to accrete intact ß2M in the form of fibrils elongated on the bead surface. Its efficiency of the ß2M removal was improved by placing the seed-immobilized beads in the middle of a continuous flow of the ß2M-containing solution as practiced in the blood circulation during the hemodialysis. Therefore, this ß2M removal system is suggested to exhibit high specificity, high binding capacity, and cost-effectiveness appropriate for eventual clinical application to remove ß2M from the blood of renal failure patients.

Nanoporous protein matrix made of amyloid fibrils of ß2-microglobulin.

Amyloid fibrils are considered as novel nanomaterials because of their nanoscale width, a regular constituting structure of cross ß-sheet conformation, and considerable mechanical strength. By using an amyloidogenic protein of ß(2)-microglobulin (ß(2)M) related to dialysis-related amyloidosis, nanoporous protein matrix has been prepared. The ß(2) M granules made of around 15 monomers showed an average size of 23.1 nm. They formed worm-like fibrils at pH 7.4 in 20 mM sodium phosphate containing 0.15 M NaCl following vigorous nondirectional shaking incubation, in which they became laterally associated and interwound to generate the porous amyloid fibrillar matrix with an average pore size of 30-50 nm. This nanoporous protein matrix was demonstrated to be selectively disintegrated by reducing agents, such as tris-(2-carboxyethyl) phosphine. High surface area with nanopores on the surface has been suggested to make the matrix of ß(2) M amyloid fibrils particularly suitable for applications in the area of nanobiotechnology including drug delivery and tissue engineering.

Seed-conjugated polymer bead for beta2-microglobulin removal at neutral pH.

beta2-Microglobulin (beta2m) is known to be a major factor for dialysis-related amyloidosis. We have studied beta2m removal through an aggregation process, which was initiated by a ligand that could catch the beta2m monomer and promote its aggregation into fibril. As a ligand, we have prepared beta2m fibril fragments and used them as a seed. The seed was coupled to PEGylated-PS beads to remove the monomeric beta2m from solution. The beta2m seed-conjugated resin effectively adsorbed the beta2m monomers with a capacity of 3.6 mg/ml via promoting their aggregation into fibrils on the resin at pH 7.4.

NMR-based characterization of a refolding intermediate of beta2-microglobulin labeled using a wheat germ cell-free system.

In patients with dialysis-related amyloidosis, beta2-microglobulin (beta2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta2-m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non-native trans-Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell-free protein synthesis and NMR techniques. The HSQC spectra of beta2-ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the beta-sheet regions especially the last half of the betaB strand and the first half of the betaE strand, both suggested to be important for amyloidogenicity, may transform beta2-m into an amyloidogenic structure.

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature.

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.

Assessment of the stability of an immunoadsorbent for the extracorporeal removal of Beta-2-microglobulin from blood.

BACKGROUND: Dialysis-related amyloidosis (DRA) is a devastating and costly condition that affects patients with end stage kidney disease. A key feature of DRA is the formation of amyloid fibrils, consisting primarily of beta2-microglobulin. Except for kidney transplantation, conventional kidney replacement therapies, which are based on nonspecific mechanisms, do not adequately address beta2-microglobulin removal. An antihuman beta2-microglobulin single-chain variable region antibody fragment (scFv) was developed to confer specificity to beta2-microglobulin removal during hemodialysis. METHODS: The scFv was immobilized onto agarose and characterized for beta2m binding capacity, thermal stability at 37 degrees C, regeneration capacity, storage conditions, and sterility. RESULTS: The beta2-microglobulin binding capacity was 1.3 mg/ml scFv gel. The immunoadsorbent is thermally stable, can be regenerated, stored short-term in 20% ethanol, lyophilized for long-term storage, and withstand process conditions similar to that of a patient's hemodialysis therapy. CONCLUSIONS: The results support further investigation of immobilized scFvs as a novel tool to remove beta2-microglobulin from blood.

Immune selection of hot-spot beta 2-microglobulin gene mutations, HLA-A2 allospecificity loss, and antigen-processing machinery component down-regulation in melanoma cells derived from recurrent metastases following immunotherapy.

Scanty information is available about the mechanisms underlying HLA class I Ag abnormalities in malignant cells exposed to strong T cell-mediated selective pressure. In this study, we have characterized the molecular defects underlying HLA class I Ag loss in five melanoma cell lines derived from recurrent metastases following initial clinical responses to T cell-based immunotherapy. Point mutations in the translation initiation codon (ATG-->ATA) and in codon 31 (TCA-->TGA) of the beta(2)-microglobulin (beta(2)m) gene were identified in the melanoma cell lines 1074MEL and 1174MEL, respectively. A hot-spot CT dinucleotide deletion within codon 13-15 was found in the melanoma cell lines 1106MEL, 1180MEL, and 1259MEL. Reconstitution of beta(2)m expression restored HLA class I Ag expression in the five melanoma cell lines; however, the HLA-A and HLA-B,-C gene products were differentially expressed by 1074MEL, 1106MEL, and 1259MEL cells. In addition, in 1259MEL cells, the Ag-processing machinery components calnexin, calreticulin, and low m.w. polypeptide 10 are down-regulated, and HLA-A2 Ags are selectively lost because of a single cytosine deletion in the HLA-A2 gene exon 4. Our results in conjunction with those in the literature suggest the emergence of a preferential beta(2)m gene mutation in melanoma cells following strong T cell-mediated immune selection. Furthermore, the presence of multiple HLA class I Ag defects within a tumor cell population may reflect the accumulation of multiple escape mechanisms developed by melanoma cells to avoid distinct sequential T cell-mediated selective events.

Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.

Single-chain antibody fragment-based adsorbent for the extracorporeal removal of beta2-microglobulin.

BACKGROUND: Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of beta2-microglobulin (beta2-m). Removal of beta2-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to beta2-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution. METHODS: An antihuman beta2-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent beta2-m in serum. RESULTS: The displayed scFv possessed a nanomolar affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed beta2-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human beta2-m. CONCLUSION: Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.