Human Immunoglobulin Light Chain Research Articles

Computational designing of the ligands of Protein L affinity chromatography based on molecular docking and molecular dynamics simulations

Protein L is a multidomain protein from Peptostreptococcus magnus with binding affinity to kappa light chain of human immunoglobulin (Ig) which is used for the purification of antibody fragments by affinity chromatography. The advances in protein engineering and computational biology approaches lead to the development of engineered affinity ligands with improved properties including binding affinity. In this study, molecular dynamics simulations (MDs) and Osprey software were used to design single B domains of the Protein L with higher affinity to antibody fragments. The modified B domains were then polymerized to ligand with six B domains by homology modeling methods. The results showed that single B domain mutants of MB1 (Thr865Trp) and MB2 (Thr847Met-Thr865Trp) had higher binding affinity to Fab compared to the wild single B domain. Also, MDs and molecular docking results showed that the polymerized Proteins L including the wild and mutated six B domains (6B0, 6B1, and 6B2) were stable during MDs and the two mutants of 6B1 and 6B2 showed higher binding affinity to Fab relative to the wild type. Communicated by Ramaswamy H. Sarma

Human Gm, Km, and Am Allotypes: WHO/IMGT Nomenclature and IMGT Unique Numbering for Immunoinformatics and Therapeutical Antibodies

Human immunoglobulin allotypes are allelic antigenic determinants (or “markers”) determined serologically, classically by hemagglutination inhibition, on the human immunoglobulin (IG) or antibody heavy and light chains. The allotypes have been identified on the gamma1, gamma2, gamma3, and alpha2 heavy chains (designated as G1m, G2m, G3m, and A2m allotypes, respectively) and on the kappa light chain (Km allotypes). Gm and Am allotypes have been one of the most powerful tools in population genetics, as they are inherited in fixed combinations, or Gm–Am haplotypes, owing to the linkage of the human IGHC genes in the IGH locus on chromosome 14. They have been very instrumental in molecular characterization of the human IGHC genes (gene polymorphisms or alleles, and IG heavy-chain structure in domains) and of the IGH locus (IGHC gene order, gene conversion, and copy number variation (CNV)). They represent a major system for understanding immunogenicity of the polymorphic IG chains in relation to amino acid and conformational changes. The WHO/IMGT allotype nomenclature and the IMGT unique numbering for constant (C) domain bridge Gm–Am and Km alleles to IGHC and IGKC gene alleles and structures and, by definition, to IG chain immunogenicity, opening the way for immunoinformatics of personalized therapeutic antibodies and engineered variants.

Sensitivity-Enhanced Multidimensional Solid-State NMR Spectroscopy by Optimal-Control-Based Transverse Mixing Sequences.

Recently, proton-detected magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy has become an attractive tool to study the structure and dynamics of insoluble proteins at atomic resolution. The sensitivity of the employed multidimensional experiments can be systematically improved when both transversal components of the magnetization are transferred simultaneously after an evolution period. The method of preservation of equivalent pathways has been explored in solution-state NMR; however, it does not find widespread application due to relaxation issues connected with increased molecular size. We present here for the first time heteronuclear transverse mixing sequences for correlation experiments at moderate and fast MAS frequencies. Optimal control allows to boost the signal-to-noise ratio (SNR) beyond the expected factor of for each indirect dimension. In addition to the carbon-detected sensitivity-enhanced 2D NCA experiment, we present a novel proton-detected, doubly sensitivity-enhanced 3D hCANH pulse sequence for which we observe a 3-fold improvement in SNR compared to the conventional experimental implementation. The sensitivity gain turned out to be essential to unambiguously characterize a minor fibril polymorph of a human lambda-III immunoglobulin light chain protein that escaped detection so far.

Germline polymorphisms and alternative splicing of human immunoglobulin light chain genes

SummaryInference of germline polymorphisms in immunoglobulin genes from B cell receptor repertoires is complicated by somatic hypermutations, sequencing/PCR errors, and by varying length of reference alleles. The light chain inference is particularly challenging owing to large gene duplications and absence of D genes. We analyzed the light chain cDNA sequences from naïve B cell receptor repertoires from 100 individuals. We optimized light chain allele inference by tweaking parameters of the TIgGER functions, extending the germline reference sequences, and establishing mismatch frequency patterns at polymorphic positions to filter out false-positive candidates. We identified 48 previously unreported variants of light chain variable genes. We selected 14 variants for validation and successfully validated 11 by Sanger sequencing. Clustering of light chain 5′UTR, L-PART1, and L-PART2 revealed partial intron retention in 11 kappa and 9 lambda V alleles. Our results provide insight into germline variation in human light chain immunoglobulin loci.

A single-chain antibody generation system yielding CAR-T cells with superior antitumor function

Cancer immunotherapy using T cells redirected with chimeric antigen receptor (CAR) has shown a lot of promise. We have established a single-chain antibody (scFv) generation system in which scFv library-expressing CAR-T cells can be screened appropriately based on their antitumor functions. A variable region library containing the variable and J regions of the human immunoglobulin light or heavy chain was fused with the variable region of a heavy or light chain encoded by an existing tumor-specific antibody to generate a new scFv library. Then, scFv library-expressing CAR-T cells were generated and stimulated with target cells to concentrate the antigen-specific population. Using this system, target-specific recognition of CAR-T cells appeared to be finely tuned by selecting a new variable region. Importantly, we have demonstrated that the newly optimized scFv-expressing CAR-T cells had better proliferation capacity and durable phenotypes, enabling superior reactivity against advanced tumors in vivo in comparison with the original CAR-T cells. Therefore, the optimization of an scFv is needed to maximize the in vivo antitumor functions of CAR-T cells. This system may allow us to adjust an immunological synapse formed by an scFv expressed by CAR-T cells and a target antigen, representing an ideal form of CAR-T-cell immunotherapy.

Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

Multiplexed CRISPR/CAS9-mediated engineering of pre-clinical mouse models bearing native human B cell receptors.

B‐cell receptor (BCR) knock‐in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one‐step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD‐GT8 60mer, a germline‐targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class‐switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.

Abstract LB-089: Targeting transferrin receptor 1 (TfR1) with the ch128.1/IgG1 antibody inhibits Epstein-Barr virus (EBV) driven lymphoproliferative growth and lymphomagenesis in immunosuppressed mice bearing human B cells

Abstract Epstein-Barr virus (EBV) is a human herpesvirus that is associated with malignancies of both B lymphocyte and epithelial cell origin. EBV is associated with the development of approximately 1.5% of all cancers worldwide, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL), Hodgkin lymphoma (HL), and post-transplant lymphoproliferative disease (PTLD). Primary infection of B cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. The TfR1 is expressed at high levels in proliferating cells including activated lymphocytes and malignant cells. We have developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has shown significant anti-tumor activity in immunosuppressed mouse models bearing human malignant B cells, including multiple myeloma and AIDS-NHL cells. In this study, we examined the effect of targeting TfR1 to inhibit EBV driven transformation of human B cells in vivo using an immunodeficient NSG mouse model. T cell-depleted human peripheral blood mononuclear cells were infected by exposure to EBV-containing supernatants from a B-lymphoblastoid cell line (B95-8). Mice were inoculated with these cells immediately following infection with EBV (day 0), or after cells were cultured for 7 days post infection prior to injection (day 7), and treated with ch128.1/IgG1 or an IgG1 isotype control antibody at 2 and 28 days post-inoculation. We monitored survival, serum levels of human cytokines, and characterized tumor-like growths in spleen and liver by assessing expression of human CD19 (marker for human B cells), EBV latent membrane protein 1 (LMP1; a marker for EBV infection), and human immunoglobulin light chain kappa or lambda (markers for monoclonality) by immunohistochemistry. Treatment with ch128.1/IgG1 significantly enhanced survival, compared to mice treated with the isotype control IgG1. Lymphoma-like cells of human B cell origin that were EBV-LMP1-positive, and often monoclonal, developed in most mice injected with EBV-infected cells (day 0 and 7) treated with the IgG1 control antibody, but only rarely in ch128.1/IgG1 treated animals. In addition, serum levels of human IL-6, IL-8, IFN-γ, CXCL13, sCD25, and sCD27, indicators of human B cell activation and proliferation, were lower in animals treated with ch128.1/IgG1. Taken together, these results show that ch128.1/IgG1 inhibited EBV driven lymphomagenesis using the NSG mouse model newly adapted to study EBV infection/transformation, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent EBV-associated B cell malignancies, including AIDS-NHL, HL, and PTLD. Citation Format: Laura E. Martinez, Tracy R. Daniels-Wells, Yu Guo, Larry I. Magpantay, Pierre V. Candelaria, Manuel L. Penichet, Otoniel Martinez-Maza, Marta Epeldegui. Targeting transferrin receptor 1 (TfR1) with the ch128.1/IgG1 antibody inhibits Epstein-Barr virus (EBV) driven lymphoproliferative growth and lymphomagenesis in immunosuppressed mice bearing human B cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-089.

Chimeric antigen receptor T cells targeting the lambda light chain of human immunoglobulin as a viable target for B cell non-Hodgkin lymphoma.

12079Background: Mantle Cell Lymphoma (MCL) is an uncommon and clinically aggressive subtype of B cell derived non-Hodgkin lymphomas (B-NHL) which continues to have poor outcomes of all B-NHLs. Che...

In vitro co-expression of human amyloidogenic immunoglobulin light and heavy chain proteins: a relevant cell-based model of AL amyloidosis

Immunoglobulin (Ig) light chain (LC) amyloidosis (AL) is characterized by the overproduction and tissue deposition of monoclonal LC in various organs and tissues. The plasma circulating monoclonal LC is believed to be the precursor of the deposited protein and in vitro studies aimed at understanding AL pathobiology have mainly focused on LC and its variable domain. While 33% of patients have free circulating monoclonal LC, ∼40% feature LC complexed to heavy chain (HC) forming a monoclonal intact Ig; the significance of free vs. bound LC in the amyloid forming pathway is unknown. To address this issue, we developed a cell-based model using stable mouse plasmacytoma Sp2/0 cells that co-express patient-derived amyloidogenic LC and HC proteins. The system was designed using amyloidogenic kappa and lambda LC, and gamma HC sequences; stable production and secretion of either free LC and/or intact Ig were accomplished by varying the LC to HC ratios. This novel cell-based system provides a relevant tool to systematically investigate LC and HC interactions, and the molecular events leading to the development of AL amyloidosis.

Ronglih Liao: Science as a Lifestyle.

Ronglih Liao studied hard as a teenager in Taiwan, but science was just one among many wide-ranging interests. At age 17, however, her focus abruptly became clear when her father was diagnosed with lung cancer, eventually leading Liao to a career in basic science with the potential to translate understanding into therapies for both rare and common cardiovascular diseases. After obtaining a chemistry degree, Liao fulfilled a promise to her late father to obtain an advanced degree overseas. For her PhD thesis at the University of Alabama Birmingham, she examined the interaction of troponin I with troponin C in bovine cardiac muscle.1 Then, Liao was recruited by 2 amazing mentors with independent laboratories and complementary expertise, Drs Judith Gwathmey and Joanne S. Ingwall, to undertake a collaborative project in cardiac physiology and myocardial energetics at Harvard Medical School. At the time, the concept that failing hearts were energy-starved was not yet accepted, and Liao deployed P31 nuclear magnetic resonance to detect decreased energy reserve in a model of dilated cardiomyopathy—baggy and sick turkey hearts—and establish its relationship to contractile performance.2 Liao set up her first independent laboratory at the Boston University School of Medicine, where she benefited from the university’s status as an internationally recognized center for the study of amyloidosis. Perfusing human immunoglobulin light chain proteins (amyloid precursors) into mouse heart, her laboratory was the first to demonstrate what the amyloidosis experts at Boston University had suspected—that amyloid light chain proteins were cardiotoxic and contributed directly to the pathogenesis and rapid progression of amyloid cardiomyopathy, independent of fibril formation and deposition.3 In 2005, Liao moved to Harvard Medical School, where she is now professor of medicine. At Brigham and Women’s Hospital, she directs the Cardiac Muscle Research Laboratory (since 2005), the Cardiovascular Flow Cytometry Core …

Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10.

BackgroundSemi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P.falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described.MethodsB lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A.ResultsSupernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10−7 M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10−9 M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10−9 M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6–6.6 mg/ml], 6.9 mg/ml (CI 5.5–8.6 mg/ml) and 9.5 mg/ml (CI 5.5–16.4 mg/ml), respectively.ConclusionThis report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.Supplementary informationThe online version of this article (doi:10.1038/gene.2014.56) contains supplementary material, which is available to authorized users.

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

Conformational Flexibility of a Human Immunoglobulin Light Chain Variable Domain by Relaxation Dispersion Nuclear Magnetic Resonance Spectroscopy: Implications for Protein Misfolding and Amyloid Assembly

The conformational flexibility of a human immunoglobulin κIV light-chain variable domain, LEN, which can undergo conversion to amyloid under destabilizing conditions, was investigated at physiological and acidic pH on a residue-specific basis by multidimensional solution-state nuclear magnetic resonance (NMR) methods. Measurements of backbone chemical shifts and amide (15)N longitudinal and transverse spin relaxation rates and steady-state nuclear Overhauser enhancements indicate that, on the whole, LEN retains its native three-dimensional fold and dimeric state at pH 2 and that the protein backbone exhibits limited fast motions on the picosecond to nanosecond time scale. On the other hand, (15)N Carr--Purcell--Meiboom--Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments of about 5-20 residues and encompassing the N-terminal β-strand and complementarity determining loop regions 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations, in the ~2-4% range. Upon acidification, the minor conformer populations increase significantly, to ~10-15%, with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. These findings provide molecular-level insights about partial protein unfolding at low pH and point to the LEN dimer dissociation, initiated by increased conformational flexibility in several well-defined regions, as being one of the important early events leading to amyloid assembly.

Lymph Node Lesion of Acute Infectious Mononucleosis in the Elderly : A Case Report

We present here lymph node lesion of infectious mononucleosis (IM) in an elderly patient. An 80-year-old Japanese woman presented with a 1 month history of left supraclavicular lymphadenopathy accompanied by high-grade fever, general fatigue and swelling in the bilateral submandibular gland region. Physical examination on admission demonstrated systemic lymphadenopathy up to 40 mm in diameter. The hemoglobin was 12.0 g/dL, white blood cell count 10.5 × 10/L (atypical lymphocytes 25%) (Fig. 1a) and platelet count 205 × 10/L. Serum protein electrophoresis demonstrated a polyclonal hyper-gglobulinemia. Measurement of serum immunoglobulin (Ig) levels demonstrated the following : IgG 4,856 mg/dL, IgA 707 mg/dL and IgM 314 mg/dL. Subsequent serologic tests for Epstein-Barr virus (EBV) showed a previous infection pattern : a viral capsid antigen (VCA) IgG titer of 4.5 (< 0.5 mg/dL), a VCA IgM titer of 0.0 (< 0.5 mg/dL), an early antigen (EA) IgG titer of 0.2 (< 0.5 mg/dL), and an EBV nuclear antigen (EBNA) titer of 1.5 (< 0.5 mg/dL). However, abnormal high titer of EBVDNA (7.6 × 10 ; normal range < 2.0 × 10) was detected in peripheral blood. Cervical lymph node biopsy was performed. Information from flow cytometry of the biopsied specimens showed a polyclonal B cell population. There was no absence of panT-cell markers. The patient treated with prednisolone, and she is currently alive without disease 14 months after disease onset. Tissue specimens were fixed in formalin solution, routinely processed and embedded in paraffin. For light microscopic examination, the sections were stained with hematoxylin-eosin (HE). Immunohistochemical studies were performed using the antigen retrieval method on the Histofine Histostainer (Nichirei Bioscience Inc, Tokyo, Japan) according to the manufacturer’s instructions. The panel of antibodies included, human immunoglobulin light chains (k and l), SP7 (CD3), 4C7 (CD5), L26 (CD20), MCS-1 (CD15), and CD30 (1G12) (Nichirei). Sections with known reactivity for antibodies assayed served as positive controls and sections treated with normal rabbit and mouse serum served as negative controls. In situ hybridization (ISH) with EBV-encoded small RNA (EBER) oligonucleotides was also performed to test for the presence of EBV small RNA in formalin-fixed paraffinembedded sections using the hybridization kit (Dako A/S, Glostrup, Denmark). DNA was extracted from paraffin-embedded sections. The variable region (CDR2 and FW3) and VDJ region (CDR3) of immunoglobulin heavy chain (IgH) gene were amplified by semi-nested PCR, using primers of FR2B, LJH and VLJH, according to a previously described method. Primers were as follows: 5’-CCGG (A/G) AA (A/G) (A/G) GTCTGGAGTGG-3’, as upstream consensus V region primer (FR2B) ; 5’-TGAGGAGACGGTGACC-3’, as a consensus J region primer (LJH) ; 5’-GTGACCAGGGT [A/C/G/T] CCTTGGCCCCAG-3’, as a consensus J region primer (VLJH). PCR products were estimated to be about 200-300 bps in length. Histologically, on low power field, the biopsied specimens demonstrated numerous lymphoid follicles with normal or atrophic germinal centers and interfollicular widening (Fig. 1b). Marked proliferation of arborizing vessels was noted in the interfollicular area (Fig. 1b). The lymphoid sinuses occasionally appeared to be compressed by the paracortical expansion. The paracortical area and medullary cords were heavily infiltrated with small and medium-sized lymphocytes, mature plasma cells, plasmacytoid cells, immature plasma cells, im-

Bioscreening of phage display antibody library and expression of a humanized single‐chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2)

Excessive expression of CTGF (connective tissue growth factor)/CCN2 has been observed in many fibrotic diseases. The inhibition of the CTGF/CCN2 by antibody has been shown to be clinically useful for the management of fibrosis. A phage display humanized single-chain Fv antibody library was screened using CTGF/C (CTGF/CCN2 C-terminal domain) as the target. A phage ELISA was performed after four rounds of biopanning, and ten positive clones were further evaluated by ELISA and were chosen for DNA sequencing. The DNA encoding scFv (single-chain variable fragment) containing a full-length variable domain fragment of heavy chain and light chain of human immunoglobulin was inserted into pET-32(a)+ vector, and the fusion protein (TrxA-scFv) containing a thrombin cleavage site was expressed mainly in soluble form. The scFv was obtained by purified fusion protein digested with thrombin and then separated from the fusion partner TrxA by gel-filtration chromatography. An immunological assay showed that the purified scFv reacted with CTGF/CCN2 in a concentration-dependent manner. The result of the cell migration assay demonstrated that the scFv at 100 ng/ml could effectively inhibit the migration of HUVEC (human umbilical-vein endothelial cells) caused by CTGF/C. The number of migratory cells was significantly decreased as compared with the negative control (1062+/-92 versus 3269+/-288, P<0.001) and the inhibition rate was 90.5%.

Efficient Agrobacterium-based transient expression system for the production of biopharmaceuticals in plants

We have recently described an efficient transient expression system mediated by Agrobacterium tumefaciens for the production of HIV-1 Nef protein in Nicotiana benthamiana plants. In order to enhance the yield of recombinant protein we assayed the effect of three gene- silencing viral suppressor proteins (P25 of Potato Virus X, P19 of Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef expression levels. Results demonstrated that AMCV-P19 gave the highest Nef yield (1.3% of total soluble protein) and that this effect was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms. Here we report additional data on the production of different heterologous proteins including human immunoglobulin heavy and light chains and a virus coat protein that demonstrate the robustness of this co-agroinfiltration expression system boosted by the AMCV-P19 gene-silencing suppressor.

Analysis of human immunoglobulin gene repertoire in NOD/scid IL2Rγ null mice engrafted with human cord blood derived hematopoietic stem cells (36.14)

Abstract The humanized NOD/scid IL2rg-/- (hNSG) and similar mouse models hold promise as testing platforms for preclinical studies. Here, we analyzed the human immunoglobulin (IG) variable heavy and light chain gene repertoire from sorted single B-cells of hNSG mice engrafted with cord blood derived hematopoietic stem cells. Unique IGHV (339) and IGLV (364, kappa and lambda) gene segments were derived from major B cell subsets identified in a largely normal B cell development pathway. However, the hNSG mice expressed elevated CD5+ mature B-1-like cells in the periphery. In mice and humans, B-1 cells are the major source of autoantibodies. Although the hNSG mice expressed a highly diverse repertoire comparable to humans, detailed genetic analysis of IG gene segments from the mature B cell subset revealed the presence of long IGH-CDR3 regions with positive charges—a hallmark for auto/polyreactivity. For functional tests, several in vitro expressed scFv-Fc IG candidates, derived from cloned heavy and light chain cognate pairs of single B cells, tested positive in an anti-nuclear antibody assay for auto/polyreactivity and/or bound to HIVgp120 in ELISA assays. The natural induction during HIV-1 infection of broadly neutralizing antibodies (BnAbs) which display autoantibody-like characteristics may be curtailed by the checkpoint control mechanisms during B cell ontogeny. We propose the hNSG mouse can be a model platform to test this hypothesis and interrogate how BnAbs are formed.

AL-Base: a visual platform analysis tool for the study of amyloidogenic immunoglobulin light chain sequences

AL-Base, a curated database of human immunoglobulin (Ig) light chain (LC) sequences derived from patients with AL amyloidosis and controls, is described, along with a collection of analytical and graphic tools designed to facilitate their analysis. AL-Base is designed to compile and analyse amyloidogenic Ig LC sequences and to compare their predicted protein sequence and structure to non-amyloidogenic LC sequences. Currently, the database contains over 3000 de-identified LC nucleotide and amino acid sequences, of which 433 encode monoclonal proteins that were reported to form fibrillar deposits in AL patients. Each sequence is categorised according to germline gene usage, clinical status and sample source. Currently, tools are available to search for sequences by various criteria, to analyse the biochemical properties of the predicted amino acids at each position and to display the results in a graphical fashion. The likelihood that each sequence has evolved through somatic hypermutation can be predicted using an automated binomial or multinomial distribution model. AL-Base is available to the scientific community for research purposes.