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When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Some misfolded proteins, e.g., immunoglobulin monoclonal free light chains (FLC), tend to form fibrils. Protein deposits in tissue may lead to amyloidosis and dysfunction of different organs. There is currently no technique allowing for the identification of FLC that are prone to aggregate. The development of such a method would enable the early selection of patients at high risk of developing amyloidosis. The aim of this study was to investigate whether silver nanoparticles (AgNPs) could be a useful tool to study the process of aggregation of FLC and their susceptibility to form the protein deposits. Mixtures of AgNPs and urine samples from patients with multiple myeloma were prepared. To evaluate the aggregation process of nanoparticles coated with proteins, UV-visible spectroscopy, transmission electron microscopy, and the original laser light scattering method were used. It has been shown that some clones of FLC spontaneously triggered aggregation of the nanoparticles, while in the presence of others, the nanoparticle solution became hyperstable. This is probably due to the structure of the chains themselves, unique protein-AgNPs interactions and perhaps correlates with the tendency of some FLC clones to form deposits. Nanoparticle technology has proven to be helpful in identifying clones of immunoglobulin FLC that tend to aggregate.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating N ε -(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

It was previously shown that several monoclonal light chains corresponding to the phagemid library of recombinant peripheral blood lymphocyte immunoglobulin light chains of patients with systemic lupus erythematosus specifically hydrolyze only myelin basic protein (MBP). Canonical enzymes usually have only one active site catalyzing some kind of chemical reaction. It was shown previously that in contrast to classical enzymes, preparations of one of the light chains (NGTA2-Me-pro-Tr) showed two optimal pH values, two optimal concentrations of metal ions, and two K m values for MBP. One protease active site of NGTA2-Me-pro-Tr was trypsin like, whereas second one was metal dependent. In this article, a search for protein sequences of NGTA2-Me-pro-Tr responsible for catalytic functions was carried out. We performed, for the first time, analysis of the homology of the protein sequence of NGTA2-Me-pro-Tr with those of several classical Zn 2+ - and Ca 2+ -dependent, as well as human serine, proteases. The analysis allowed us to identify the protein sequences of NGTA2-Me-pro-Tr responsible for serine-like activity, the binding of MBP, and chelation of metal ions and catalysis directly. The data obtained are summarized using hypothetical models of the structure of the two active centers of a very unusual light chain of antibodies (Abs). The findings obtained may be very important for understanding possible structure of active centers of very unusual light chain of Abs possessing several enzymatic activities., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call "Crystal Kappa", enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions., Competing Interests: RL, SA, ZD, CH, NRK, AP, JW and SA are, or were at the time of the work described here, employees of Eli Lilly & Co. Eli Lilly & Co. provided support in the form of salaries for the authors and research funding. Eli Lilly & Co. also approved the publication of this work. Eli Lilly & Co. has filed a patent application related to the work described here. The employment of the authors by Eli Lilly & Co does not alter our adherence to PLOS ONE policies on data sharing and materials.

KappaSelect is an affinity medium that specifically binds to the constant region of the kappa light chain (LC). Obviously, KappaSelect can be used to separate antibody species containing the kappa LC constant region from those lacking that region. However, it is not clear whether this resin can readily separate species containing one kappa LC constant region from those containing two kappa LC constant regions although the former are assumed to bind weaker than the latter. In this work, we demonstrated that antibody species with two kappa LC constant regions binds to the KappaSelect resin much tighter than species with only one kappa LC constant region. Consequently, these two species can be readily separated using this resin. This information not only enriches our knowledge with KappaSelect but also has important practical value. In addition to the sample case used in the current study, there are other cases in which the target protein contains one kappa LC constant region whereas a byproduct contains two kappa LC constant regions. Our finding provides a convenient means for removing such two kappa LC binding site containing byproduct in these cases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m / z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m / z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1 k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m / z .

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains., Competing Interests: Competing interest statement: G.D.Y. is a board member, employee, and shareholder of Regeneron Pharmaceuticals, Inc. Other authors are employees and shareholders of Regeneron Pharmaceuticals, Inc.

Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved β-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Determination and quantification of serum free light chains (FLC) is essential for the diagnosis and monitoring of patients with monoclonal gammopathies. Currently, the Freelite assay (The Binding Site, United Kingdom) and the N Latex FLC assay (Siemens Healthineers, Germany) are available for FLC determination. Data concerning stability of FLC following long-term storage are limited. Therefore, the aim of the present study is to investigate the stability of FLC in frozen samples determined by the N latex FLC assay., Methods: One hundred eighty-eight serum samples from 47 patients with monoclonal gammopathies were analyzed at the beginning and after long-term storage (-20°C, 193 - 568 days). Serum free light chain kappa (κFLC) and lambda (λFLC) concentrations were measured, and the corresponding kappa/lambda (κ/λ FLC) ratios were calculated. All samples were analyzed with the N latex FLC assay on a single BNII System., Results: Comparison analyses between fresh and stored samples revealed very strong correlations for the determination of κFLC (r = 0.993), λFLC (r = 0.998) and the calculated κ/λ FLC ratio (r = 0.997). Median percentage changes of κFLC (-0.5%) and λFLC (-0.8%) measurements and the calculated κ/λ FLC ratios (-3.7%) were within the calculated analytical imprecision of the FLC assay. Changes of κFLC (r = 0.17, p = 0.02) and λFLC (r = 0.28, p < 0.01) concentrations and of the κ/λ FLC ratio (r = 0.18, p = 0.01) over time were very weak, but statistically significant., Conclusions: Serum free light chains in serum samples are sufficiently stable following long-term frozen storage and are therefore suitable to be measured with the N Latex FLC assay.

The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rabbit antibodies show unique structural characteristics in that kappa chains have an inter-domain disulfide bond between the variable and constant domains. Here we characterized this disulfide bond from physicochemical viewpoints both in stability and affinity. It was revealed that the disulfide bond contributed to the thermal stability of the antibody, but the affinity and mechanism of antigen recognition was not altered by the mutation. The present result expands the understanding of how rabbit antibodies with kappa light chains gain affinity under characteristic mechanism to gain thermal stability, and would give suggestions for the methods to artificially stabilize antibody molecules.

Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody-antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (V H H) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody-ligand complex and lowers its aggregation temperature to favor crystallization. The V H H crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The production of antibodies is accompanied by a slight excess of synthesis of κ and λ immunoglobulin light chains; small amounts of them are released in the peripheral blood and can also be found in various body fluids, such as synovial fluid, cerebrospinal fluid, urine and saliva. They are rapidly filtered by the glomerulus and >99% are reabsorbed from the cells of the proximal convoluted tubule, making them present in the urine in only trace amounts. The production of an excess of protein without a reason or a specific function in a biological system is rare. Free light chains, considered for years a waste product of Ig synthesis, are currently known to be very active molecules, able to bind antigens as well as whole immunoglobulin and helping to develop specific antibody affinity. The ability of free light chains to activate mast cells and then become an active part of the pathogenic mechanisms of chronic inflammatory diseases has increased interest in their clinical use, both as an attractive therapeutic target or as a biochemical marker of disease evolution or remission. This is an overview of relevant scientific interest that immunoglobulin light chains κ and λ have attracted over the years, a report on the progress in knowledge about their structure and function, with a special focus on their biological meaning and potential clinical utility in different diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use., (© 2017 The Protein Society.)

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (C κ ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-C κ , [-5]proPSA-C κ , and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-C κ fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a + B cells were more abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM + B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In patients with rheumatic diseases, reliable markers for determining disease activity are scarce. One potential parameter is the level of immunoglobulin free light chains (FLCs), which is known to be elevated in the blood of patients with certain rheumatic diseases. Few studies have quantified FLCs in urine, a convenient source of test sample, in patients with different rheumatic diseases. We carried out a retrospective analysis of patients with rheumatic disease attending the University hospital of Goettingen, Germany. Subjects were included if they had urine levels of both κ and λ FLCs available and did not have myeloma. Data regarding systemic inflammation and kidney function were recorded, and FLC levels were correlated with inflammatory markers. Of the 382 patients with rheumatic disease, 40.1 % had chronic polyarthritis, 21.2 % connective tissue disease, 18.6 % spondyloarthritis and 15.7 % vasculitis. Elevated levels of κ FLCs were found for 84 % of patients and elevated λ for 52.7 %. For the patients with rheumatoid arthritis, FLCs correlated with C-reactive protein (κ, r = 0.368, p < 0.001; λ, r = 0.398, p < 0.001) and erythrocyte sedimentation rate (κ, r = 0.692, p < 0.001; λ, r = 0.612, p < 0.001). Patients being treated with rituximab displayed FLC levels similar to those of the reference group. There were clear elevations in both κ and λ FLCs in patients with rheumatic disease, but not in κ/λ ratio. The correlation between FLCs and inflammatory markers in patients with rheumatoid arthritis demonstrates their potential for predicting disease activity.

Capillary electrophoresis/immunosubtraction (CE/IS) is a simple method for detecting and immunotyping serum or urine monoclonal proteins. To our knowledge, there are no previous reports of the use of CE/IS for characterizing patients with Immunoglobulin light chain (AL) amyloidosis, and there are no convincing data available to compare CE/IS with serum immunofixation electrophoresis (IFE) and free light chain (FLC) assay. The aim of this study was to evaluate the clinical utility of CE/IS in patients with AL amyloidosis as a diagnostic accuracy study. This study included 50 patients with AL amyloidosis (17 newly diagnosed and 33 undergoing treatment). Serum IFE identified monoclonal proteins in 15/50 (30%) of all cases and in 7/17 (41%) of newly diagnosed cases. CE/IS identified monoclonal proteins in 16/50 (32%) of all cases and in 7/17 (41%) of newly diagnosed cases. The FLC assay detected an abnormal ratio of kappa and lambda light chains in 26/50 (52%) of all cases and in 15/17 (88%) of newly diagnosed cases. IFE and CE/IS combined with FLC assay identified monoclonal proteins more sensitive than IFE alone and CE/IS alone, in newly diagnosed patients (p = 0.002 and 0.002, respectively) and in patients undergoing treatment (p = 0.031 and 0.016, respectively). CE/IS is an acceptable alternative to IFE.

It was shown previously that approximately 30% ± 5% of antibodies against myelin basic protein (MBP) and the DNA of patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS) possess catalytic activities that play an important negative role in the pathogenesis of MS and SLE. An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). The clones were expressed in E. coli in a soluble form. MLChs were purified by metal chelating chromatography followed by FPLC-gel filtration. The activity of one MLCh (NGTA1-Me-pro) was inhibited only by EDTA, and it efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four known immunodominant sequences containing cleavage sites of MBP only in the presence of several different metal ions. An unexpected result was obtained: NGTA1-Me-pro demonstrated two pH optima, two optimal concentrations of Me 2+ ions, and two K m values for MBP. The protein sequence of NGTA1-Me-pro, having two metalloprotease active centers, has homology with several mammalian metalloproteases. Recently, it was shown that one other MLCh possesses serine-like and metalloprotease activity. The principal possibility of the existence of MLChs with several different active centers is unexpected, but very important for the further understanding of unknown possibilities for immune systems and the biological functions of antibodies.

In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.

For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection.

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

Polyclonal antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose. The fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27kDa). The clones were expressed in Escherichia coli in a soluble form; MLChs were purified by metal-chelating chromatography followed by gel filtration. In mammalians, there are serine proteases and metalloproteases. These and many other enzymes usually have only one active site and catalyze only one chemical reaction. In contrast to canonical proteases, one MLCh (NGTA2-Me-pro-ChTr) efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four immunodominant sequences containing cleavage sites of MBP. The proteolytic activity of MLCh was efficiently inhibited only by specific inhibitors of serine-like (phenylmethanesulfonylfluoride, PMSF) and metalloproteases (EDTA). It was shown that MLCh possess independent serine-like and metal-dependent activities. The principal existence of monoclonal antibodies with two different proteolytic activities is unexpected but very important for the further understanding of at present unknown biological functions of human antibodies., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Monoclonal antibodies (mAb) have had tremendous success in treating a variety of cancers over the past twenty years. Yet despite their widespread clinical use, which includes treatments for haematological malignancies, there are still no approved mAb therapies for multiple myeloma (MM). This is likely to change within the next few years with a number of mAb therapies being assessed in late stage clinical trials, most notably, the anti-CS-1 mAb, elotuzumab, and the anti-CD38 mAb, daratumumab, which are currently being evaluated in Phase III clinical trials for MM. In this review, we will discuss the preclinical and clinical development of MDX-1097, a Phase II candidate which targets cell membrane-associated kappa immunoglobulin free light chains expressed on the surface of MM cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analog, was generated by covalently conjugating two engineered [des-His1, Ala129Cys]FGF21 molecules to a nontargeting human IgG1 κ scaffold. The pharmacokinetics (PK) of PF-05231023 after i.v. and s.c. administration was evaluated in rats and monkeys using two enzyme-linked immunosorbent assays with high specificity for biologically relevant intact N termini (NT) and C termini (CT) of FGF21. Intact CT of FGF21 displayed approximately 5-fold faster systemic plasma clearance (CL), an approximately 2-fold lower steady-state volume of distribution, and at least 5-fold lower bioavailability compared with NT. In vitro serum stability studies in monkeys and humans suggested that the principal CL mechanism for PF-05231023 was degradation by serum proteases. Direct scaling of in vitro serum degradation rates for intact CT of FGF21 underestimated in vivo CL 5-fold, 1.4-fold, and 2-fold in rats, monkeys, and humans, respectively. The reduced steady-state volume of distribution and the bioavailability for intact CT relative to NT in rats and monkeys were compatible with proteolytic degradation occurring outside the plasma compartment via an unidentified mechanism. Human CL and PK profiles for intact NT and CT of FGF21 were well predicted using monkey single-species allometric and Dedrick scaling. Physiologically based pharmacokinetic models incorporating serum stability data and an extravascular extraction term based on differential bioavailability of intact NT and CT of FGF21 in monkeys improved accuracy of human PK predictions relative to Dedrick scaling. Mechanistic physiologically based pharmacokinetic models of this nature may be highly valuable for predicting human PK of fusion proteins, synthetically conjugated proteins, and other complex biologics., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3&#95;LV1DE and P2224KV3&#95;LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3&#95;LV1DE and P2224KV3&#95;LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

Because DNase antibodies are cytotoxic, enter the nucleus and cause DNA fragmentation inducing cell death by apoptosis, they can play an important role in the pathogenesis of different autoimmune pathologies and especially systemic lupus erythematosus (SLE). The interesting goal of catalytic antibodies research is not only to study a possible biological role of such antibodies, but also to develop in future new human and animal therapies that use the advantages offered by abzymes. An immunoglobulin κ light chain library from SLE patients was cloned into a phagemid vector. Phage particles displaying recombinant monoclonal antibody light chains (MLChs) capable of binding DNA were isolated by affinity chromatography on DNA-cellulose. Sixteen of the 46 MLChs efficiently hydrolyzed DNA; one MLCh (approximately 27-28kDa) was expressed in Escherichia coli and purified by metal chelating and gel filtration. MLCh NGK-1 was electrophoretically homogeneous and demonstrated a positive answer with mouse IgGs against light chains of human antibodies after western blotting. SDS-PAGE in a gel containing DNA demonstrated that the MLCh hydrolyzes DNA and is not contaminated by canonical DNases. The DNase MLCh was activated by several metal ions. The protein sequence of the DNase MLCh has homology with mammalian DNases I and shares with them several identical or similar (with the same side chain functionality) important amino acid residues, which are necessary for DNA hydrolysis and binding of Mg(2+) and Ca(2+) ions. The affinity of DNA for this first example of a MLCh (K(M) = 0.3 microM) was 150- to 200-fold higher than for human DNase I., (© The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km  = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases., (Copyright © 2013 John Wiley & Sons, Ltd.)

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

An efficient and potentially scalable technology was evaluated to control the ice nucleation step of the freezing process for a model monoclonal antibody formulation and the effect on process performance and quality attributes of the final lyophilized product was compared with the conventional shelf ramping method of freezing. Controlled ice nucleation resulted in uniform nucleation at temperatures between -2.3 and -3.2 °C while uncontrolled nucleation resulted in random nucleation at temperatures between -10 and -16.4 °C. The sublimation rate (dm/dt) during primary drying was higher in the controlled nucleation cycle (0.13 g/h/vial) than in the uncontrolled nucleation cycle (0.11 g/h/vial). This was due to the formation of larger ice crystals, leading to lower product resistance (Rp) and 19% reduction in the primary drying for the controlled nucleation cycle. Controlled ice nucleation resulted in lyophilized cakes with more acceptable appearance, no visible collapse or shrinkage and decreased reconstitution times compared with uncontrolled nucleation. There were no observed differences in the particle size, concentration (A280 nm) and presence of aggregates (A410 nm) between the two nucleation cycles when the lyophilized cakes were reconstituted. These were confirmed by SEC and protein A-HPLC analyses which showed similar peak shapes and retention times between the two cycles. However, uncontrolled nucleation resulted in cakes with larger specific surface area (0.90 m(2)/g) than controlled nucleation (0.46 m(2)/g). SEM images of the lyophilized cakes from uncontrolled nucleation revealed a sponge-like morphology with smaller pores while cakes from controlled nucleation cycle revealed plate-like structures with more open and larger pores. While controlled nucleation resulted in a final product with a higher residual moisture content (2.1±0.08%) than uncontrolled nucleation (1.62±0.11%), this was resolved by increasing the secondary drying temperature., (Published by Elsevier B.V.)

In our search of new biomarkers for multiple sclerosis (MS), we aimed to characterize the immunoglobulin (Ig) free light chains (FLC) in patients' cerebrospinal fluid (CSF) and serum, and to evaluate the diagnostic utility of FLC monomer-dimer patterns for MS. FLC were analyzed by Western blotting and mass spectroscopy. CSF and serum samples were examined for the presence of oligoclonal Ig bands by a conventional laboratory test for MS. Three distinct pathological FLC monomer-dimer patterns, typical of MS but not of other neurological diseases, were revealed. In 31 out 56 MS patients the highly increased CSF levels of κ monomers and dimers were demonstrated. In 18 MS patients, the increased κ-FLC levels were accompanied by highly elevated λ dimers. Five MS cases showed no significant elevation in κ-FLC, but they displayed abnormally high λ dimer levels. The intensity of the immunoreactive FLC bands was measured to account for κ and λ monomer and dimer levels and their ratios in the CSF and serum. Combined usage of different FLC parameters allowed the determination of the appropriate FLC threshold values to diagnose MS. The developed method showed higher sensitivity and specificity (96% and 90%, respectively), as compared to those of the conventional OCB test (82% and 70%, respectively). Our study highlights the role of the differential analysis of monomeric and dimeric κ- and λ-FLC for the precise diagnosis of MS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF(Fbs2) E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.

Background: Anti-cytokine autoantibodies (auto-Abs) are ubiquitous both in patients suffering from infectious, inflammatory and autoimmune diseases and in healthy individuals. Particularly anti-IFN-γ auto-Abs are shown to be elevated in blood of multiple sclerosis (MS) patients., Objective: The aim of present study was to investigate whether repertoires of anti-IFN-γ auto-Abs differ in MS patients and healthy donors., Methods: Using phage display technique we have compared repertoires of the genes encoding anti-IFN-γ single-chain variable fragments selected from MS and naïve phage display libraries., Results: The panel of anti-IFN-γ auto-Abs selected from MS library includes (i) 'fetal' auto-Abs, encoded by the VH6-1 gene segment and the combination proximal D segments with distal JH segments; (ii) naïve auto-Abs; (iii) affinity maturated antibodies; and (iv) abnormal single-domain antibodies. Meanwhile, the panel of anti-IFN-γ auto-Abs selected from naïve library mainly contains the naïve antibodies. Moreover, the overall antibody repertoire of MS library is skewed compared to the overall repertoire of naïve library and also contained the antibodies carrying a 'fetal' VH6 domain and the ratio of κ and λ chains was reversed., Conclusions: These results suggest existence of a special mechanism or trigger that provides for reconstitution of the immune system in MS.

A comparative transcription analysis of Ig κ-light chains (IGK) of the cattle breeds Holstein Friesian (HF), German Black Pied (GBP), German Simmental (GS) and Aubrac (A) revealed three alleles coding for two putative allotypic variants of IGKC. The amino acid residues p.Asp100Asn and p.Thr116Ala were located at the outer edge of the constant domain as demonstrated by homology-based modelling. Alleles were distributed in unequal frequencies within the breeds examined. While cattle breeds HF, GS, and A possessed all alleles and allotypic variants, GBP exhibited alleles encoding allotypic variant IGKC(a) . All three IGKJ segments were detected in 320 sequences analysed. IGKJ1 was combined predominantly with IGKC. The ORF2 of IGKJ2 was detected for the first time on transcriptional level., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

For the purpose of investigating the carboxy terminus distribution of immunoglobulin κ light chain in Aκ amyloid deposits in tissue sections, we examined the immunostaining pattern of Aκ amyloidosis with conventional rabbit clonal antibody against peptide derived from the C-terminal sequence of human κ light chain. This antihuman kappa light chain clone II (clone H16-E) reacted with the adjacent region of the C terminus of the κ light chain constant region in SPOT analysis. Immunohistochemically, this antibody reacted with amyloid deposits in all 18 cases of Aκ amyloidosis. In 15 cases, this antibody reacted with amyloid deposits almost uniformly. In this study, we demonstrated for the fi rst time that the peptides adjacent to the carboxy terminus of immunoglobulin κ light chain or full-length κ light chain were constituents of Aκ amyloidosis, and these molecules were distributed uniformly in almost all cases of Aκ amyloidosis in tissue sections.