Light Chain Variable Region Genes Research Articles

Generation of a Phage Display Chicken Single-Chain Variable Fragment Library.

Phage-displayed antibody fragment libraries can be constructed using essentially any species that is easily immunized, as long as the immunoglobulin variable region gene sequences are known. This protocol describes the procedures for the generation of a phage-displayed chicken single-chain variable fragment (scFv) library after immunization with a target antigen. Briefly, the rearranged heavy chain variable region (V H ) genes and the λ light chain variable region (V λ ) genes are amplified separately and are linked through two separate PCR steps to give the final scFv genes. The genes are then cloned into pComb3XSS to generate the phage display chicken scFv library, which can then be used for test and final library ligations.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.

Using Next Generation Sequencing to Identify Trackable Clonotypic Sequences for Minimal Residual Disease Testing in AL Amyloidosis

Introduction: Recent studies have shown that deeper free light chain responses are associated with improved outcomes in patients with systemic light chain (AL) amyloidosis, yet the optimal long-term goal remains unclear.(N Engl J Med 2018; 378:518-528) Unlike multiple myeloma, where the role of minimal residual disease (MRD) testing is well established, in AL amyloidosis the methods to best identify MRD, and whether achieving MRD would benefit disease outcomes, warrant further investigation. (Blood Cancer J. 2021 Jun 21;11(6):117; PLoS ONE 15(7): e0235713. https://doi.org/10.1371/journal.pone. 0235713) In AL amyloidosis, the toxic misfolded oligomers and amyloid deposits are both due to clonal immunoglobulin light chains and cause organ damage and death. Hence, the tracking of clonotypic light chain sequences could present a promising approach for detecting the maintenance of profound therapeutic responses. Our study aimed to assess the frequency with which next generation sequencing (NGS) detects light-chain clones consistent with the patient's clonal light-chain isotype and to determine whether the light-chain sequences identified were encoded by light-chain variable region genes considered AL-related.(Journal of Clinical Oncology 2019 37:15_suppl, 8010; Blood (2017) 129 (3): 299-306; PLoS One 2022 Feb 25;17(2):e0264407) Methods: Ninety patients diagnosed with AL amyloidosis between September 2016 and July 2023 at Tufts Medical Center and University of California San Francisco were included. Bone marrow samples from the 90 patients were sent to Adaptive Biotechnologies Inc. (Seattle, WA) for initial clone identification using the clonoSEQ Assay. IMGT/BlastSearch database (Version 1.2.1) was used to identify the clonotypic immunoglobulin light chain variable domains detected by the clonoSEQ assay. Samples were then divided in to two groups based on the consistency of paraprotein isotype with the trackable sequences, and Mann-Whitney U test was used to compare the difference of the numbers of clonotypic sequences. Results: Of the 90 patients with bone marrow samples sent for clone identification, 85 (94.4%) had at least one trackable clonotypic sequence, with a median number of 3 and a maximum of 11. Of the 85 patients with trackable sequences, 67 (78.8%) had an AL paraprotein isotype consistent with the detected clonotypic sequence isotype, 48 with light chain and 19 with heavy chain isotypes. Compared to the inconsistent ones, patients who had consistency in paraprotein and trackable sequences exhibited higher numbers of clonotypic sequences ( P = 0.001). With respect to marrow plasma cell infiltrations, both consistent and inconsistent groups had a median of 15% marrow plasma cells with no statistically significant difference between them by two-tailed Mann-Whitney. Of note, consistent with the restricted pattern of germline gene use in AL, the 48 patients also used AL-related variable region Ig light chain genes; the variable region light chain genes identified were AL-related and included IGKV1-33 (8 identified sequences), IGKV4-1 (4), IGKV1-16 (3), IGKV1-5 (1), GKV1-13 (1), IGLV6-57 (8), IGLV3-21 (6), IGLV1-44 (5), IGLV3-1 (4), IGLV1-40 (3), IGLV2-14 (3), IGLV1-51 (2). Conclusion: In patients with AL amyloidosis, the ClonoSEQ assay can identify a sequence consistent with the patient's light or heavy chain isotype almost 80% of the time. Of those cases in which a clonal light chain sequence was consistent with the patient's isotype, the genes identified were from the well described AL-related light chain variable region gene repertoire. These findings indicate that the size of the AL clone does not affect this outcome and suggest that other factors related to the adaptive immune dysregulation of AL or to the multiplex PCR process may be involved. ClonoSEQ can identify trackable clonotypic light chain sequences that are AL-related 56% of the time in patients with AL, a finding that may be relevant to patients with myeloma.

Novel neutralizing mouse-human chimeric monoclonal antibodies against the SARS-CoV-2 receptor binding domain.

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.

Abstract 1325: Novel recombinant rabbit monoclonal antibodies for cancer biology research

Abstract Recombinant monoclonal antibodies (rAbs) are superior tools for cancer biology research as they offer distinct advantages over standard polyclonal and hybridoma-generated monoclonal antibody reagents. Recombinant technology produces antibodies that can be defined at the primary sequence level and are characterized by consistent performance and supply. These rAb features are in stark contrast to traditional polyclonal and hybridoma-generated monoclonal antibodies, whose lack of dependability has been widely documented as a primary reason for data irreproducibility plaguing biomedical research. In an effort to respond to this challenge, GeneTex has established a recombinant antibody production platform combined with more rigorous validation protocols. The goal is to develop reliable recombinant rabbit monoclonal antibodies for reproducible performance in various experimental applications utilized routinely by cancer biologists. GeneTex’s rAb production protocol is based on the approach described by Starkie et al. (2016). This involves a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and isolate antigen-specific IgG+ memory B cells obtained from an immunized rabbit. The heavy and light chain variable region genes from selected cells are PCR-amplified and cloned into plasmids to produce full-length heavy and light chains for a single IgG. These constructs are subsequently introduced together into mammalian cells for expression, meaning that natural pairing of the chains is maintained. This manufacturing strategy allows application-specific testing (e.g., for western blot (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC/IF), etc.) of clones during the antibody production process. Validation of antibodies is performed inhouse and, when possible, in relationships with academic or industry/pharma institutions where ideal samples and reagents are frequently more available. This recombinant monoclonal antibody production platform has created a series of well-validated antibodies against many important targets for cancer biology research. This includes reagents to study tumor immunobiology (e.g., PD-L1), hormone receptors (e.g., ER alpha and androgen receptor variants like ARV7), oncogenic RAS protein mutants (e.g., RAS G12D), transcription factors (e.g., NRF2), and epithelial-mesenchymal transition (EMT) (e.g., E-cadherin), among many others. Extensive validation for multiple applications was performed, with knockdown or CRISPR-based knockout cell lysates employed when possible to establish specificity. In summary, GeneTex is utilizing a FACS-based recombinant rabbit monoclonal antibody production approach to generate and thoroughly validate antibody reagents that will hopefully facilitate reproducible cancer biology research. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Ping Kuan Hsieh, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel recombinant rabbit monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1325.

Hyper N-Glycosylated SEL1L3 As B-Cell Receptor Autoantigen of Primary Vitreoretinal Lymphoma

Chronic antigenic stimulation of B lymphocytes through their B-cell receptor (BCR) is thought to play an important role in the genesis of B-cell lymphomas. SAMD14/neurabin-I has been described as auto-antigenic target of primary central nervous system lymphoma (PCNSL) BCRs. Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress to PCNSL. Besides manifestation in an immunologically privileged site, PVRL and PCNSL share frequent recurrent genetic alterations like MYD88 and CD79B mutations. Both PVRL and PCNSL are considered being related to the MCD subtype of DLBCLs and may depend on chronic BCR signaling through antigenic stimulation. To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed BCRs from PVRL patients as antibody fragments (Fabs) and tested for specific binding against common antigens. All cases with corresponding BCR sequences were previously reported by Belhouachi et al. (Blood Adv. 2020;4(7):1357-1366). For 20 of 55 PVRL BCR sequences both immunoglobulin heavy and light chain variable region (V) genes were available. DNA clones comprising an ApaLI restriction site, the complete light chain V gene, a κ- or λ-constant region gene, a ribosome binding site, a signal sequence, the heavy chain V gene and a BstEII restriction site were ordered from GeneCust® (GeneCust, 5690 Ellange, Luxembourg) in a pUC57 vector. DNA fragments were cloned in front of a γ-1 constant region gene and a Histidine-tag into a modified pCES-1 vector for expression as Fabs. PVRL-BCRs were screened on protein macroarrays representing clones of UniPEx human cDNA expression libraries expressed in E. coli (Bioscience, Dublin, Ireland). SEL1L3 was identified as the auto-antigenic target of the B-cell receptors in 3 out of 20 PVRL cases. None of the PVRL Fabs bound to LRPAP1 (common BCR antigen of mantle cell lymphomas), RpoC (Moraxella catarrhalis bacterial antigen and target of nodular lymphocyte-predominant Hodgkin lymphoma cells) or galectin-3 (member of the lectin family). SEL1L3 is a 1132 amino acid long protein with multiple glycosylation and phosphorylation sites (uniprot.org). This is especially important since previously identified BCR antigens from different lymphoma entities showed post-translational modifications such as hyper-N-glycosylation or hypo-phosphorylation. Furthermore, SEL1L3 is highly expressed in the pigmented layer of the retina. Western Blot analysis of the blood of a PVRL patient with serum antibodies against SEL1L3 showed an increased molecular weight of SEL1L3 as compared to SEL1L3 of controls. After treatment with N-acetyl-β-d-glucosaminidase or PNGase F, this difference in molecular weight disappeared, indicating a hyper N-glycosylation of SEL1L3 in PVRL patients with SEL1L3-reactive BCRs as trigger for its' immunogenicity. SEL1L3 induced proliferation of aggressive lymphoma cell lines transfected with SEL1L3-reactive BCRs. Moreover, the BCR binding epitope of SEL1L3 (aa 560 - 585) conjugated to a truncated form of Pseudomonasaeruginosa exotoxin A, killed exclusively lymphoma cells expressing BCRs with the respective reactivity. Additionally, BCRs of a locally treated PVRL patient with serum antibodies against SEL1L3 were cloned from biopsies of the vitreous body at initial diagnosis and of a lymphoma manifestation in the breast at relapse. Both BCRs showed binding to SEL1L3 suggesting a continued dependence of the BCR on antigen stimulation. These results suggest an important role of chronic antigenic stimulation by the post-translationally modified auto-antigen SEL1L3 in the genesis of a subgroup of patients with PVRL, might give an explanation for the selective tropism of PVRLs and provide the basis for a new treatment approach with high specificity.

Detection of Clonal Plasma Cells in POEMS Syndrome Using Multiparameter Flow Cytometry

Detection of Clonal Plasma Cells in POEMS Syndrome Using Multiparameter Flow Cytometry

Identification of clonal immunoglobulin λ light-chain gene rearrangements in AL amyloidosis using next-generation sequencing

Amyloid light-chain (AL) amyloidosis is caused by deposition of abnormally folded clonal immunoglobulin (Ig) light chains made by malignant plasma cells in the bone marrow (BM), leading to multiorgan dysfunction. However, little is known of the factors that regulate the organ tropism of amyloid deposition in this disease. We aimed to identify the clonal composition of Igλ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using next-generation sequencing. Based on our definition of the clonal IGLV rearrangement (dominant clone >2.5%, dominant cluster >5%), we identified clonal IGLV in 33 of 38 patients with AL amyloidosis (86.8%), 6 of 9 with monoclonal gammopathy of undetermined significance (67%), and 7 of 7 with multiple myeloma (100%). The clones in AL amyloidosis were significantly smaller than those in multiple myeloma (p < 0.01) but comparable to those in monoclonal gammopathy of undetermined significance. Importantly, in patients with AL amyloidosis, the difference in involved and uninvolved free light chains was not correlated with the clonal size of BM plasma cells in our repertoire analysis using NGS. In summary, the clonal composition of IGLV genes in the BM was successfully identified in most patients with AL amyloidosis using NGS. The clonal size of plasma cells in the BM is small, and small malignant clones of plasma cells may secrete free light chi and cause light chain depositions in AL amyloidosis.

IGVL gene region usage correlates with distinct clinical presentation in IgM vs non-IgM light chain amyloidosis

AbstractPatients with immunoglobulin M (IgM) light chain (AL) amyloidosis have a distinct clinical presentation compared with those with non-IgM amyloidosis. We hypothesized that differential immunoglobulin light-chain variable region (IGVL) gene usage may explain the differences in organ involvement, because IGVL usage correlates with organ tropism. IGVL usage was evaluated by mass spectrometry of amyloid deposits (IgM, n = 45; non-IgM, n = 391) and differed across the 2 groups. In the λ family, LV2-08 (13% vs 2%; P < .001) and LV2-14 (36% vs 10%; P < .001) usage was more common in IgM vs non-IgM amyloidosis, whereas LV1-44 (0% vs 10%; P = .02) and LV6-57 (2% vs 18%; P = .004) usage was less common. In the κ family, there was a trend toward higher KV4-01 (11% vs 4%; P = .06) usage in IgM amyloidosis. IGVL usage correlated with disease characteristics/organ tropism. LV2-14 (more common in IgM amyloidosis) has historically been associated with peripheral nerve involvement and lower light chain burden, which were more frequent in IgM amyloidosis. LV1-44 (less common in IgM), associated with cardiac involvement, was less frequent in IgM patients. LV6-57 (less common in IgM) is associated with t(11;14), which was less frequent in IgM patients. In conclusion, IGVL gene usage differs in patients with IgM vs non-IgM amyloidosis and may explain the distinct clinical presentation.

Preparation of anti-GPC3 single chain antibody for targeted detection of hepatocellular carcinoma

Glypican-3 (GPC3) is a key member of Glypican family and plays an important role in the development, angiogenesis and metastasis of hepatocellular carcinoma (HCC). Most HCC overexpresses GPC3, but GPC3 is hardly detected in normal adult liver and benign liver lesions, so it is regarded as a highly specific diagnostic marker and an ideal therapeutic target for HCC. In this study, we cloned the heavy and light chain variable region gene from the monoclonal antibody targeted to GPC3 screened in the previous stage, and linked it with a segment of flexible peptide (Linker) to obtain the single chain antibody against GPC3. The single chain antibody gene was cloned into vector for prokaryotic expression and purified to obtain high purity protein. Detection shows that the single-chain antibody produced by us has the same binding activity with the full-length antibody, and can accurately target the tumor site of Huh7 tumor-bearing model mice after coupling Cy5.5 fluorescence, suggesting that the single-chain antibody has the potential to realize multi-directional liver cancer precise surgical navigation under the guidance of a probe.

Generation of mycobacterial lipoarabinomannan-specific monoclonal antibodies and their ability to identify mycobacterium isolates.

The World Health Organization has recommended commercial urine-sourced lipoarabinomannan (LAM) detection as a tool for screening HIV patients with suspected TB, but more sensitive immunodetection assays would help to identify HIV-negative TB patients. Here, we aimed to develop novel rabbit monoclonal antibodies (mAbs) against LAM for immunodetection purposes. Rabbits were immunized with cell-wall components from the Mycobacterium tuberculosis (Mtb) H37Rv strain. An immune single-chain fragment variable (scFv) phage display library was generated. The scFv mAbs to LAM were identified through ELISA screening. The light and heavy chain variable region genes from the selected clones were sequenced. Vectors containing the full-length light and heavy chains were constructed and co-expressed in 293T cells to generate whole IgG antibodies. The performances and binding characteristics of the mAbs against purified LAM from M.tb H37Rv, multiple mycobacteria species (M.tb H37Rv, M. bovis and non-tuberculous mycobacteria (NTM) strains), and mycobacteria clinical isolates (Mtb and NTM isolates) were determined using various immunoassay methods. We obtained five rabbit mAbs against LAM, four of which had high sensitivities (100pg/ml) and affinities (1.16-1.73×10-9M) toward LAM. They reacted with M.tb H37Rv, M. bovis, and slow-growing NTM, but not with rapid-growing NTM. Similar results were obtained with mycobacterium isolates, where 96% of the Mtb isolates and 90% of the M. avium-intracellulare isolates were successfully identified. The novel rabbit LAM-specific mAbs performed well at recognizing LAM from slow-growing pathogenic mycobacteria, which support their future clinical application.

Immunoglobulin Variable Gene Region (IGVL) Usage Correlates with Distinct Clinical Presentation in IgM Versus Non-IgM Light Chain Amyloidosis

Introduction: The clinical presentation and frequency of organ involvement are quite distinct in patients with IgM amyloidosis compared to patients with non-IgM amyloidosis, with less heart involvement and more nerve, soft tissue and lung involvement in IgM patients. It has been observed that immunoglobulin light chain variable region (IGVL) gene and gene family of the plasma cell clone impact phenotypic manifestations in AL amyloidosis, particularly organ tropism. (Kourelis et al. Blood. 2017;129). Given the variability in clinical features amongst patients with IgM vs. non IgM AL amyloidosis, we hypothesized that IGVL gene usage would be different between these two groups. Methods: Patients with newly diagnosed IgM and non-IgM amyloidosis seen at our institution from 01/2006 to 12/2015 were identified. IGVL gene usage was assessed by liquid chromatography tandem mass spectrometry (LC/MS/MS) as previously described. (Vrana et al Blood 2009;Dasari et al J of Proteome Res; Kourelis et al. Blood. 2017). Mass spectrometry data for IGVL analysis was obtained from clinically available data. LC/MS/MS was performed on available archival specimens when such data was not available clinically. Results: Patients with newly diagnosed amyloidosis who had sufficient mass spectrometry data on fat aspirate or tissue biopsy for IGVL gene usage identification were included (IgM AL, N=44 and Non-IgM AL, N=391). Clinical Features: As expected, differences were noted in baseline characteristics of patients with IgM vs. non-IgM AL amyloidosis. Patients with IgM amyloidosis were older (68 vs. 64 years, p=0.04) and more likely to have kappa as the involved light chain (34% vs. 25%, p=0.2), though the difference did not reach statistical significance. IgM amyloidosis patients had lower difference in involved and uninvolved free light chains (dFLC) (15.5 vs. 25.8 mg/dL, p=0.01) and lower NTProBNP levels (median: 1987 vs. 2655 pg/mL, p=0.02) compared with non-IgM amyloid patients. Presence of t(11;14) was less common by FISH (30% vs. 48%, p=0.1), though the difference did not reach statistical significance. Patterns of organ involvement were different, with heart involvement being less frequent in IgM patients (64% vs. 76%), while peripheral nerve (30% vs. 18%, p=0.05), soft tissue (41% vs. 21%, p=0.005) and lung involvement (7% vs. 3%, p=0.1) were more common in patients with IgM vs. non-IgM AL amyloidosis. There was no difference in liver (9% vs. 15%), kidney (50% vs. 51%) GI (23% vs. 25%) or autonomic nervous system involvement (18% vs. 14%). IGVL Gene Usage: As shown in Figure 1, IGVL usage differed across the two groups. In the lambda group, LV2-08 (14% vs. 2%, p&lt;0.001) and LV2-14 (36% vs. 10%, p&lt;0.001) were more common in IgM amyloid patients, while LV1-44 (0% vs. 10%, p=0.02) and LV6-57 (2% vs. 18%, p=0.004) were less common in the IgM group. There was also a trend towards less frequent involvement of LV3-01 (2% vs. 12%, p=0.07) in IgM amyloid patients. In the kappa group, KV4-01 (11% vs. 4%, p=0.04) was more common in patients with IgM amyloidosis. Correlation Between IGVL Gene usage and Clinical Features: IGVL gene usage correlated with disease characteristics and differences in the clinical presentation of patients across the two groups. LV2-14 was more commonly seen in IgM amyloid patients (above). It has been previously associated with a higher frequency of peripheral nerve involvement (Kourelis et al. Blood 2017), which was more frequent in IgM group. LV2-14 is also associated with low dFLC levels, which were noted to be lower in the IgM AL cohort. LV1-44 has been associated with higher likelihood of cardiac involvement. LV1-44 was seen less frequently in the IgM amyloid patients, who also had lower rates of cardiac involvement. LV6-57 has been historically associated with higher rates of t(11;14). It was less common in IgM AL patients, who also had lower rates of t(11;14). Conclusions: IGVL gene usage differed significantly in patients with IgM vs. non-IgM amyloidosis, correlating with differences in disease characteristics and organ involvement. This is the first study to our knowledge to report IGVL gene usage differences in IgM vs. non-IgM AL amyloidosis. Differences in IGVL gene usage may explain the distinct clinical presentation seen in IgM amyloidosis, the biology of which has so far remained poorly understood. *SS and SD contributed equally Disclosures Dasari: The Binding Site: Patents &amp; Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Dispenzieri:Alnylam: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Pfizer: Research Funding; Janssen: Consultancy; Akcea: Consultancy; Intellia: Consultancy. Murray:The Binding Site: Patents &amp; Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding. Gertz:Prothena: Honoraria; Alnylam: Honoraria; Ionis: Honoraria; Janssen: Honoraria; Spectrum: Honoraria, Research Funding; Celgene: Honoraria.

Identification of Clonal Immunoglobulin λ Light-Chain Gene Rearrangements in AL Amyloidosis Using Next Generation Sequencing

[Introduction] AL amyloidosis is caused by the deposition of abnormally folded clonal immunoglobulin (IG) light chains (LCs, λ:κ = 3:1) made by malignant plasma cells in the bone marrow (BM), which leads to multi-organ dysfunction, often involving the heart, kidney, liver, skin, and nerves. However, little is known about what regulates organ tropism of amyloid deposition in this disease. In addition, no study has analyzed the repertoire of IG germlines of plasma cells in the BM in AL amyloidosis using next generation sequencing (NGS). In this study, we aimed to identify the clonal composition of IG λ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using NGS. [Material and method] BM cells were obtained at diagnosis from 38 patients with AL amyloidosis and those with other plasma cell disorders: multiple myeloma (MM, n = 7), and monoclonal gammopathy of undetermined significance (MGUS, n = 11) with λ-type monoclonal paraprotein. Seven normal control (NC) patients had either immune thrombocytopenia or malignant lymphoma without BM invasion. Genomic DNA was extracted from the BM mononuclear cells preserved in LABO Banker1 or BM clots in O.C.T compound using QIAamp DNA Blood Mini kit. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction using a 5′ primer for the IGLV1/2 framework 3 (FR3) region and 3′ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were performed using an Illumina MiSeq sequencer and then analyzed by an open-source program called Vidjil. All subjects provided written informed consent to participate in the study, in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of the Chiba University Graduate School of Medicine and Japanese Red Cross Medical Center. [Results] Clinical and laboratory features of 38 patients with AL amyloidosis were as follows: primary AL amyloidosis (n = 31); 15 and 20 patients had cardiac and renal dysfunctions, respectively, and secondary AL amyloidosis with MM (n = 7); 4 and 1 patient had cardiac and renal dysfunctions, respectively. In patients with AL amyloidosis, the median plasma cell count in BM aspirate smears was 3.3% (0.1%-50.4%), and the median difference in involved and uninvolved light chains (dFLC) was 104.5 mg/L (28.5mg/L -2673.3mg/L). Representative results of the Vidjil analysis in NC, MGUS, AL amyloidosis, and MM are shown in Figure 1. The most abundant IGLV gene accounted for not &gt;1% of the reads, and there was no dominant germline in NC samples. Therefore, we defined the dominant clone as &gt;1% of IG germlines in plasma cells. According to this definition, clonal IG germlines were found in 27 of 31 patients with primary AL amyloidosis (87%), 5 of 7 with secondary AL amyloidosis (71%), 7 of 7 with MM (100%), and 8 of 11 with MGUS (73%). However, the size of clones in AL amyloidosis (median 3.1%, 0.38%-14.3%) was significantly smaller than that in MM (median 17.8%, 2.2%-17.9%) (P&lt;0.001), and similar to that in MGUS (median 3.8%, 0.4%-32.0%). Importantly, in patients with AL amyloidosis, the dFLC and involved/uninvolved FLC ratio was not correlated with the clonal size of plasma cells in our repertoire analysis using NGS, suggesting that small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Regarding IGLV germline usage, IGLV1-51 was the most frequent repertoire in AL amyloidosis with heart dysfunction (7 of 16 cases) and renal dysfunction (7 of 21 cases). No relationship between the IGLV germlines and organ tropisms was observed. [Conclusion] We successfully identified the clonal composition of IGLV genes in the BM of most patients with AL amyloidosis using NGS, according to the differences in the V and J region recombination and CDR3 sequences using the Vidjil program. In AL amyloidosis, the clonal size of plasma cells in the BM is small and small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Figure 1 Disclosures Suzuki: Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential implications for immune checkpoint blockade and neoantigen-based immunotherapies

BackgroundDiffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma.MethodsTo determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens’ immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry.ResultsSHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell–like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell–like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM.ConclusionsThese results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

Generation of single-chain Fv antibody fragments against Mu-2-related death-inducing gene in Escherichia coli.

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.

Long-term and high dose dexamethasone injection decreases the expression of Immunoglobulin Heavy (Light) Chain Variable Region Genes (IGH(L)Vs) in the mouse spleen

Glucocorticoid hormones have been widely used in clinical practice as potent anti-inflammatory and immunosuppressive agents. However, the underlying mechanisms of how they work remain unaddressed. Here, we used RNA-set to profile spleen gene expression in adult mice after consistent intraperitoneal injection of dexamethasone. We identified 13565 genes in control (injected with 0.9% NaCl) and 13702 genes in dexamethasone-injected group, 12920 genes were expressed in both, but 645 genes were identified only in control and 782 genes were identified only in the dexamethasone group. In the dexamethasone-injected group 101 and 67 genes were down-and up-regulated, respectively. Among these, 129 were coding genes, 19 were identified as non-coding genes or pseudogenes, and the remaining 20 were TEC (to be experimentally confirmed) genes. Gene ontology (GO) and KEGG pathway analysis revealed that the cytokine-cytokine receptor interaction pathway was highly enriched in these differentially expressed genes, and that a majority of the 129 identified coding genes were involved in immune system and cell adhesion-associated processes. Moreover, systemic lupus erythematosus, renin-angiotensin system, fat digestion and absorption, and glycerolipid metabolism pathways were significantly affected in the dexamethasone-treatment group. No obvious signaling pathway was enriched in the control group. Additionally, 20 immunoglobulin heavy or light chain variable region genes (IGH(L)Vs) were down regulated in the dexamethasone-injected group. IGH(L)Vs encode the variable region of immunoglobulin heavy chain and determine the diversity and specificity of antibodies. We were unable to determine the function of the 19 non-coding genes with differential expression following dexamethasone treatment. Our findings indicate that the expression of IGH(L)Vs and non-coding genes play an important role in the anti-inflammatory and immunosuppressive effects of dexamethasone and could be developed as potential agents in clinical practice.

Clonal immunoglobulin λ light-chain gene rearrangements detected by next generation sequencing in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a rare plasma cell dyscrasia characterized by polyneuropathy, organomegaly, endocrinopathy, extravascular fluid overload, M protein, and a myriad of skin changes. The pathogenesis is poorly understood, but monoclonal plasma cells are λ-restricted and these immunoglobulin λ light chain variable (IGLV) region genes are derived from only two germlines, either IGLV1-44 or 1-40. Here we analyzed the clonal IGLV gene rearrangements of genomic DNA samples of bone marrow mononuclear cells using next-generation sequencing (NGS) to understand the clonal composition of IGLV genes in patients with POEMS syndrome (n = 30). The dominant IGLV gene rearrangement of POEMS syndrome-specific germline sequences were significantly increased in 11 POEMS patients (36.7%; IGLV1-44: n = 9, IGLV1-40: n = 2). In some cases, IGLV gene rearrangement clone was not detected as significant increase but was detected using cDNA samples by heteroduplex (HD) analysis and Sanger sequencing, suggesting that the quite small number of monoclonal plasma cells may produce large quantity of mRNA of monoclonal proteins. However, significant increase of dominant clone sizes was not directly linked to the initial disease status. On the other hand, in cases with significantly increased dominant clones, they decreased and increased accompanying with disease remission and relapse. These data demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

Clarifying immunoglobulin gene usage in systemic and localized immunoglobulin light-chain amyloidosis by mass spectrometry

AbstractThe goal of this study was to investigate the frequency of use of light-chain variable region (IGVL) genes among patients with systemic (ALS) and localized (ALL) amyloidosis and to assess for associations between IGVL gene usage and organ tropism. We evaluated clinic charts from 821 AL patients seen at the Mayo Clinic who had bone marrow, fat pad, and solid organ tissue samples typed by liquid chromatography tandem mass spectrometry (LC-MS). We identified 701 patients with ALS and 120 with ALL. Overall, we were able to identify an IGVL gene in 87 (72%) patients with ALL and 573 (82%) patients with ALS. When compared with ALL, LV6-57 was more common, whereas KV3-20 and heavy-chain codeposition were less common in ALS. In this large series of ALS, characteristics particular to specific genotypes became apparent. LV6-57 patients were more likely to have renal involvement and to harbor a translocation 11;14. LV3-01 patients were less likely to have advanced cardiac disease and renal involvement. LV2-14 patients were more likely to have peripheral nerve involvement, an intact circulating immunoglobulin, and lower circulating dFLC. LV1-44 patients were more likely to have cardiac involvement. KV1-33 patients had more liver involvement and higher circulating dFLC. Finally, KV1-05 was associated with inferior overall survival but not independently of cardiac stage. IGVL gene usage appears to provide clues about disease pathophysiology and tissue tropism. LC-MS is a high-throughput and low-resource technique that can be used to identify IGVL gene from clinical tissue specimens.

Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM.