Heavy Chain Variable Region Research Articles

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Epigallocatechin Gallate (EGCG) Promotes the Immune Function of Ileum in High Fat Diet Fed Mice by Regulating Gut Microbiome Profiling and Immunoglobulin Production.

This study aimed to investigate the regulatory effect of epigallocatechin gallate (EGCG) on the composition of the gut microbiome, the transcriptomic profiling of ileum, and their interplay in high fat diet (HFD) induced obese mice. Intragastric administration of EGCG to C57BL/6J mice for 14 consecutive weeks remarkably decreased HFD induced excessive fat deposition (p < 0.001), and the increment of serum TG, TC, HDL-C (p < 0.05), as well as improved glucose tolerance (p < 0.001). EGCG shifted the gut microbiota mainly by elevating the relative abundance of Parasutterrlla, Bacteroides, and Akkermansia (p < 0.01), decreasing that of norank_f_Erysipelotrichaceae, unclassified_f_Ruminococcaceae, Anaerotruncus, Roseburia, norank_Lachnospiraceae, and Lachnospiraceae_UCG_006 (p < 0.01) at the genus level. In addition, EGCG affected the transcriptomic profiling of ileum, and the differentially expressed (DE) genes after HFD or/and EGCG treatment were mostly enriched in the immune reaction of ileum, such as the GO term of “immune effector process” and “phagocytosis, recognition.” Furthermore, the KEGG category of “immune diseases,” “immune system,” and “infection diseases: bacterial” were commonly enriched by the DE genes of the two treatments. Among those DE genes, 16 immunoglobulins heavy chain variable region encoded genes (Ighvs) and other immunity-related genes, such as complement component 2 (C2), interferon-induced transmembrane protein 1 (Iftm1), polymeric immunoglobulin receptor (pigR), and alanyl aminopeptidase (Anpep), were highly correlated with the shifted microbes in the gut (p < 0.05, absolute r > 0.5). Overall, the results suggested that EGCG ameliorated the HFD induced metabolic disorder mainly by regulating gut microbiome profiling and the immunoglobulin production of ileum, while the genes expressed in the ileum, especially Ighvs, C2, Iftm1, pigR, and Anpep, might play important roles in coordinating the immunity of mice regarding the gut microbes and the host interactions.

Primary Pulmonary Mucosa-associated Lymphoid Tissue Lymphoma with the High Expression of IgG4.

This is the first report describing primary pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma with the high expression of IgG4. The histological findings were compatible with the diagnostic criteria for MALT lymphoma and IgG4-related respiratory disease (IgG4-RRD). An unfixed sample for Southern blotting was not obtained since computed tomography findings showed multiple lung cysts, which is rare in patients with MALT lymphoma. However, polymerase chain reaction using paraffin sections showed the clonality of the immunoglobulin heavy chain variable region gene rearrangement, confirming a diagnosis of MALT lymphoma. This is an instructive case in which primary pulmonary MALT lymphoma was histologically compatible with IgG4-RRD.

Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

It is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

Repertoire of Rearranged Immunoglobulin Heavy Chain Genes in Russian Patients With B-Cell Lymphoproliferative Diseases

IntroductionImmunoglobulin heavy chain variable region (IGHV) repertoire narrowing could be an evidence for the involvement of a limited set of antigens in the development of lymphomas. For chronic lymphocytic leukemia (CLL) the existence of more than 200 subgroups of tumor IGHV antigen-binding sites, so called “stereotypical” antigen receptors (SAR) has been shown. For others lymphomas the possibility of SARs is also suggested. The aim of this study is to compare the tumor IGHVs and possible SARs in various B-cell malignancies in Russia and other countries. Materials and MethodsThe study included samples of 1800 CLL patients, 52 patients with mantle cell lymphoma, 48 patients with hairy cell lymphoma and 37 patients with splenic marginal cell lymphoma. The nucleotide sequences of the IGHV genes were determined according to ERIC protocol. ResultsIn CLL most common IGHV genes were IGHV1-69, IGHV1-2, IGHV3-30 and IGHV4-34. The most common SARs were CLL#1, CLL#6, CLL#2, CLL#3. In MCL the most common genes were IGHV4-34, IGHV3-21, IGHV3-23. In 5 MCL patients CDR3 sequences were identified matching definitions of a stereotyped. In the half of SMZL patients was identified gene IGHV1-2. Other IGHV genes were much less common. Two pairs of SMZL patients have motives similar to each other. In HCL IGHV repertoire was the most variable, no trends for antigen receptor stereotypy were observed. It was found that SARs are highly disease-specific both at the level of nucleotide and amino acid sequences. ConclusionOur results suggest that antigens crucial for the pathogenesis of B-cell malignancies could be disease-specific. Further studies on extended samples of non-CLL patients concerning the role of SARs in pathogenesis of these diseases may also contribute to the development of new diagnostic and prognostic markers.

Leukemic Variant of Mantle Cell Lymphoma: Clinical Presentation and Management.

This review summarizes the unique presentation and management of the leukemic variant of mantle cell lymphoma (LV-MCL, also referred to as non-nodal MCL) and highlights the biologic and clinical differentiation from classical mantle cell lymphoma (cMCL) in biomarker expression, clinical features, prognosis, disease course, and treatment. Several studies have evaluated the gene expression profile of mantle cell lymphoma, differentiating LV-MCL from cMCL. The typical immunophenotypic profile is CD5-positive, SOX 11-negative, CD23-low, CD200-low, and cyclin D1 overexpressed. LV-MCL commonly has mutated immunoglobulin heavy chain variable region genes. Data on treatment of LV-MCL is limited to retrospective analyses; the ideal treatment for these patients is unknown although many have a clinically indolent, asymptomatic presentation and often may be observed for an extended period without active treatment. LV-MCL is a clinically and biologically distinct entity. Clinically, it must be distinguished from chronic lymphocytic leukemia and cMCL. Future prospective, randomized clinical trials are required to optimize management, define the initial treatment, and appropriately sequence treatment modalities.

Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BackgroundApproximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception.MethodsHeavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb [“Human Contraception Antibody” (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model.FindingsHCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue.InterpretationHCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception.FundingThis research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

The Prognostic Significance of the Complete IgH Rearrangement Pattern Using the BIOMED-2 Protocol in Diffuse Large B-Cell Lymphoma

▪Introduction: In several B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, the somatic hypermutation (SHM) status of immunoglobulin heavy chain (IgH) variable region (VH) significantly correlates with clinical outcome. Diffuse large B-cell lymphoma (DLBCL) usually contains a number of VH SHMs, with considerably variable frequency. However, there have been few reports about the association between VHSHM frequency and clinical outcome in DLBCL.The BIOMED-2 multiplex PCR protocol is one of the standardized methods for detecting IgH gene rearrangements, and has been used to prove clonality in B-cell lymphomas. VH SHMs in the germinal center prevent proper annealing of the applied PCR primers, rendering clonal VH rearrangements undetectable by this protocol. Thus, the complete IgH rearrangement pattern produced by the BIOMED-2 protocol is expected to reflect the frequency of VH SHMs. In this study, we investigated the prognostic impact of complete IgH rearrangement pattern on patients with DLBCL, and analyzed the correlation between complete IgH rearrangement pattern and VH SHM frequency by Sanger sequencing.Materials and Methods: We retrospectively identified 386 patients diagnosed with de novo DLBCL that received anthracycline-based immunochemotherapy at Kobe City Medical Center General Hospital from January 2005 to June 2015. We excluded 83 patients because fresh samples were not available for PCR analysis, and a total of 303 patients were enrolled. Based on the BIOMED-2 protocol, complete IgH rearrangement was detected by three PCR reactions using three VH forward primer sets: framework region (FR) 1, FR2, and FR3. The primary endpoint was 5-year overall survival (5-yr OS). The secondary endpoint was 5-year progression-free survival (5-yr PFS). OS and PFS were estimated using the log-rank test. Multivariate analysis using the Cox proportional hazard ratio was performed for 5-yr OS. For VH sequencing analysis, the cryopreserved PCR products of 42 available samples were sequenced directly using both forward and reverse primers, and for each sequenced sample, the percentage of VH identity to the closest germline gene was calculated, using the immunoglobulin gene database (IMGT/V-QUEST).Results: Of the 303 patients enrolled (median age: 69 years old), 183 patients (60.3%) had advanced stage disease and 148 (48.8%) had a high-intermediate or high International Prognostic Index (IPI) score. The median follow-up was 60.6 months (2.0-133.8 months). Complete IgH rearrangement was detected in 176 (58.1%), 172 (56.8%), and 113 (37.2%) of patients using the FR1, FR2, and FR3 primer sets, respectively. In total, 80 patients (26.4%) were positive for all three PCR reactions, 82 (27.1%) for two, 57 (18.8%) for one, and 84 (27.7%) had completely negative results. We compared the 5-yr OS and PFS among these subgroups (Figure 1), and observed that the probability of survival decreased with accumulation of positive PCR results. As shown in Figure 2, both the 5-yr OS and PFS were significantly worse for patients with all three reactions positive for complete IgH rearrangement (OS: 54.2% vs 73.2% p=0.002; PFS: 34.4% vs. 59.3%, p<0.001). In multivariate analysis adjusted by IPI score, complete IgH rearrangement detected by all three PCR reactions was significantly correlated with poor prognosis (hazard ratio: 2.13; 95% CI: 1.38-3.26; p<0.001).This classification also allowed subgroups with unfavorable prognosis to be identified, even among patients with a high-intermediate or high IPI score (5-yr OS: 28.0% vs. 62.9%, p<0.001). Moreover, VH sequencing analysis of 42 available samples confirmed that patients with complete IgH rearrangement in all three PCR reactions had significantly lower VH gene SHMs than other groups (median value of VH identity; 91.9% vs 84.0%, p<0.001).Conclusion: Complete IgH rearrangement detected by all three primer sets in the BIOMED-2 PCR protocol was associated with poor prognosis in patients with DLBCL. Combined with the additional sequencing analysis, this suggests that the lower levels of VH SHMs may be associated with unfavorable prognosis in DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Validated Risk Model for Overall Survival in Relapsed/Refractory Chronic Lymphocytic Leukemia Applicable to Patients Treated with Novel Therapies and Standard of Care

Introduction:Several prognostic models for overall survival (OS) in chronic lymphocytic leukemia (CLL) have been developed, but none were derived in a relapsed/refractory (R/R) population or in the context of novel therapies. We therefore used a comprehensive model-building approach to derive and validate a novel risk model for OS in a population of previously treated patients (pts) with CLL who received therapy in randomized phase 3 trials of ibrutinib (IBR) vs standard of care (SOC) and idelalisib (IDELA) vs SOC.Methods:In collaboration with Janssen Pharmaceuticals, Gilead Sciences, and Pharmacyclics, the analysis included pts from 5 multicenter randomized phase 3 trials (n=1866): IBR plus bendamustine-rituximab (BR) vs placebo plus BR (HELIOS: NCT01611090), IBR vs ofatumumab (RESONATE: NCT01578707), IDELA plus BR vs placebo plus BR (Study 115: NCT01569295); IDELA plus rituximab vs placebo plus rituximab (Study 116: NCT01539512); and IDELA plus ofatumumab vs ofatumumab (Study 119: NCT01659021). All pts had a diagnosis of CLL, were previously treated, and required treatment.The model-building dataset(n=969) included pts from HELIOS and RESONATE, and was randomly assigned to the training dataset (n=727) and internal-validation dataset (n=242). The external-validation dataset was an independent pooled cohort from studies 115, 116, and 119 (n=897).We applied univariate and multivariate analyses (MVA) to 28 candidate clinical, laboratory and molecular/genetic prognostic factors to derive the risk model in the training dataset. The primary endpoint was OS. We assessed the need for separate models for novel therapies by interactions with treatment in univariate analyses.We evaluated different cutoffs for dichotomized covariates and for definition of risk groups after the final factors were selected. We fit a Cox regression on the training, internal-validation ,and external-validation datasets using the risk categories as the covariate to test the difference among the groups, and calculated the C-statistic as a measure of discrimination.Results:We proceeded with the development of a single risk score for all pts with R/R CLL, as only one potential prognostic factor (deletion 13q, interaction p value <0.10) demonstrated a differential effect necessitating separate models. Six factors were independently prognostic in the multivariate analysis of OS (p<0.05): β2-microglobubulin (B2M), lactose dehydrogenase (LDH), hemoglobin (HGB), immunoglobulin heavy chain variable region (IGHV) mutational status, number of prior therapies, and time from initiation of last therapy.We excluded number of prior therapies from the final multivariate model, reasoning that its optimal cutoff might change with advances in CLL therapies. IGHV did not remain significant (p=0.0592) in MVA of 5 remaining prognostic factors. Notably, deletion 17p was not independently prognostic for OS.The final risk model consisted of 4 prognostic factors: B2M ≥5 mg/L, LDH elevated, HGB <110 mg/L for women or <120 mg/L for men, and time from initiation of last therapy <24 months. Because hazard ratios of these factors were similar (range, 0.55-0.68) with overlapping confidence intervals, 1 point was assigned to each factor. The resulting score separated pts into low (score 0-1), intermediate (score 2-3), and high risk (score 4) groups.Our risk model was prognostic for OS in the training dataset (log-rank p<0.0001; C-statistic 0.74 [95% CI 0.60-0.85]) and was confirmed in the internal-validation dataset (log-rank p<0.0001; C-statistic 0.79 [95% CI 0.56-0.97]), as well as in the external-validation dataset (log-rank p<0.0001; C-statistic 0.71 [95% CI 0.59-0.81]).Conclusions:We present the first validated risk model for OS in relapsed/refractory CLL in the era of targeted therapies. Our model consists of 4 readily available factors (B2M, LDH, HGB, time from initiation of last therapy) and identifies 3 prognostic groups with significantly different OS. This model reliably identifies high risk pts. Investigators can address this unmet need by designing prospective trials targeting these pts. [Display omitted] DisclosuresDarif:Janssen: Employment, Equity Ownership. Londhe:Janssen: Employment, Equity Ownership. Xing:Gilead: Employment, Equity Ownership. Huang:Gilead: Employment. Lin:Gilead: Employment. Byrd:Acerta Pharma: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics: Research Funding; Janssen: Research Funding. Furman:Pharmacyclics: Consultancy, Honoraria; Genentech: Consultancy; Gilead: Consultancy; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy. Hallek:Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Hillmen:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Research Funding. Jones:Sunesis: Other: Institutional research funding; Abbvie, Pharmacyclics, Genentech, Gilead, Janssen, Merck, and Acerta: Other: Institutional research funding; Genentech, Abbvie, Pharmacyclics, Gilead, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sharman:Gilead Sciences, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding; Genentech: Research Funding. Ferrante:Janssen: Employment, Equity Ownership. Reddy:Pharmacyclics/Abbvie: Employment, Equity Ownership. Dreiling:Gilead Sciences, Inc.: Employment. Adewoye:Gilead: Employment. Dubowy:Gilead: Employment. Howes:Janssen R&D: Employment, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Equity Ownership. James:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Zelenetz:Celgene: Consultancy; Amgen: Consultancy.

T Cells Expressing Anti-B-Cell Maturation Antigen (BCMA) Chimeric Antigen Receptors with Antigen Recognition Domains Made up of Only Single Human Heavy Chain Variable Domains Specifically Recognize Bcma and Eradicate Tumors in Mice

Multiple myeloma (MM) is an almost-always incurable malignancy of plasma cells. Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T-cell signaling domains. CAR T cells have been shown to have substantial activity against leukemia and lymphoma, which has generated great interest in developing CAR T-cell therapies for MM. CAR T cells targeting B-cell maturation antigen (BCMA) have been shown to have substantial activity against MM (Ali et al. Blood. 2016. 128(13). 1688-1700). Improvements in CAR T-cell therapies for MM are needed. One possible limitation of CAR T-cell therapies is generation of recipient anti-CAR immune responses. Recipient anti-CAR immune responses have been reported in patients on clinical trials of anti-CD19 CARs (Turtle et al. Journal of Clinical Investigation. 2016. 126 (6). 2123-3138). Recipient anti-CAR immune responses could potentially develop against murine antibody variable region domains that are included in the single-chain variable fragments (scFvs) of most CARs. In addition, recipient anti-CAR immune responses could develop against the artificial linker domain in the scFv or against junctions between different CAR components.We designed CARs with antigen-recognition domains of single fully-human heavy-chain-only variable domains (FHVH). The FHVH CARs do not contain a light chain variable region domain or a linker. The fully-human variable region, lack of a linker, and the minimized number of junctions should all lead to reduced immunogenicity for the FHVH antigen-recognition domain compared with a standard scFv.We constructed CARs with 4 different FHVH domains designated FHVH32, FHVH33, FHVH74, and FHVH93. We designed CARs with each of these FHVH domains along with a CD8-alpha hinge and transmembrane domain, either a CD28 or a 4-1BB costimulatory domain, and a CD3-zeta T-cell activation domain. DNA encoding these CARs was ligated into a gamma-retroviral vector, and primary human T cells were transduced with gamma-retroviruses encoding each of the CARs. We tested the T cells for CAR expression and a variety of T-cell functions. We assessed T-cell surface CAR expression by staining with phycoerythrin-labeled BCMA protein. T cells from 3 MM patients were transduced with genes encoding each of the CD28-containing CARs. The following mean percentages of T cells expressed each CAR after transduction: FHVH32-CD828Z (47.3%), FHVH33-CD828Z (49.6%), FHVH74-CD828Z (51.3%), and FHVH93-CD828Z (46.6%). The mean T-cell surface CAR expression of the 11D5-3-CD828Z CAR containing a standard murine scFv with a heavy chain variable domain and a light chain variable domain connected by a linker was 60.4%. Importantly, we found that each of the FHVH CARs specifically recognized BCMA in functional assays. Specific interferon gamma production by the CARs is shown in the Table. We also demonstrated BCMA-specific tumor-necrosis factor-alpha release, cytotoxicity, and proliferation by T cells expressing each of the 4 FHVH CARs containing CD28. We have previously reported clinical anti-myeloma responses with infusions of T cells expressing the 11D5-3-CD828Z CAR (Ali et al. Blood. 2016. 128(13). 1688-1700). T cells expressing FHVH CARs with CD28 domains, especially FHVH33-CD828Z and FHVH74-CD828Z, had BCMA-specific functions including cytokine release that were equal to or superior to those of T cells expressing 11D5-3-CD828Z (Table). We have demonstrated that human T cells expressing either FHVH33-CD828Z or FHVH74-CD828Z can eliminate established tumors of BCMA-expressing human RPMI8226 cells from immune-deficient mice.We also assessed FHVH CARs with 4-1BB costimulatory domains. Of the 4-1BB CARs, FHVH33-CD8BBZ CAR had the highest percentage of T cells with surface CAR expression (mean 52.1%, n=3). T cells expressing FHVH33-CD8BBZ exhibited BCMA-specific cytokine-release, degranulation, cytotoxicity, and proliferation.In summary, we have designed, constructed, and tested a novel type of CAR with an antigen-recognition domain made up of only single fully-human heavy-chain variable region domains. These CARs specifically recognized BCMA in vitro, and they eradicated tumors from mice. The simple, fully-human antigen-recognition domains in these CARs offer a potential advantage of decreased immunogenicity compared to other CAR designs. [Display omitted] DisclosuresTrinklein:TeneoBio: Employment. Buelow:TeneoBio: Employment. Kochenderfer:Bluebird bio: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.; Kite Pharma: Research Funding.

Increase of DNA Damage and Alteration of the DNA Damage Response in Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia

The increase of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in the pathogenesis of monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL). Immunofluorescence microscopy of γH2AX and 53BP1 was performed for the analysis of DNA double-strand breaks (DSB) in CD19+ lymphocytes of 6 healthy individuals, 6 MBL patients, 21 CLL patients and in cells of the CLL cell line HG-3. In addition, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was used for the analysis of the DNA damage response (DDR). As compared to the γH2AX foci levels in healthy individuals (0.1 focus per CD19+ lymphocyte ± 0.0; mean ± standard error of mean) increased γH2AX foci levels were detected in MBL patients (0.3 focus per CD19+ lymphocyte ± 0.1), CLL patients (1.3 foci per CD19+ lymphocyte ± 0.3) and in cells of the cell line HG-3 (1.0 focus per cell). Particularly high γH2AX foci levels were observed in immunoglobulin heavy-chain variable-region (IGHV)-unmutated, 11q-deficient, 17p-deficient and TP53 -mutated CLL. Co-localization of γH2AX and 53BP1 indicated promotion of 53BP1-mediated nonhomologous end-joining (NHEJ) repair mechanisms at sites of DSB. Absent expression of p-CHK1 and p-TP53 suggested impaired G2/M checkpoint control and impaired apoptosis signaling in MBL and CLL. In summary, our results provide evidence for a continuous increase of DNA damage across the spectrum from MBL to CLL in conjunction with an impaired DDR. Particularly high γH2AX foci levels were detected in IGHV -unmutated, 11q-deficient, 17p-deficient and TP53 -mutated CLL. Moreover, abundant γH2AX/53BP1 foci suggested a critical role for (in)effective NHEJ repair mechanisms at sites of DSB in MBL and CLL.Figure legendsFig. 1. Analysis of γH2AX foci in MBL and CLL patient samples. (A) Fluorescence microscopic image of γH2AX foci (green, Alexa 488) in nuclei (blue, DAPI) of the sample CLL8. (B) γH2AX foci levels in CD19+ lymphocytes of healthy individuals, MBL and CLL patients. (C) γH2AX foci levels in CLL samples according to CD38 and ZAP-70 expression. (D) γH2AX foci levels in CLL samples according to the immunoglobulin heavy-chain variable-region mutational status. (E) γH2AX foci levels according to cytogenetic risk categories. The categories were defined by (1) harboring a normal karyotype, (2) a 13q deletion as sole aberration, (3) a 12q trisomy but not an 11q or 17p deletion, (4) an 11q deletion but not a 17p deletion and (5) a 17p deletion. (F) γH2AX foci levels according to gene mutation categories. The categories were defined (irrespective of the karyotype) by harboring (1) no mutations in SF3B1, NOTCH1 and TP53, (2) a SF3B1 mutation but not a TP53 mutation, (3) a NOTCH1 mutation but not a TP53 or SF3B1 mutation and (4) a TP53 mutation (irrespective of other mutations). Note: Each dot in the graphs B - F represents the mean γH2AX foci level of one sample. MBL, monoclonal B-cell lymphocytosis; CLL, chronic lymphocytic leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Bruton’s Tyrosine Kinase in CLL

Targeting the B-cell receptor signaling pathway through BTK inhibition proved to be effective for the treatment of chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. Covalent BTK inhibitors (BTKis) led to an unprecedented improvement in outcome in CLL, in particular for high-risk subgroups with TP53 aberration and unmutated immunoglobulin heavy-chain variable-region gene (IGHV). Ibrutinib and acalabrutinib are approved by the US Food and Drug Administration for the treatment of CLL and other B-cell lymphomas, and zanubrutinib, for patients with mantle cell lymphoma. Distinct target selectivity of individual BTKis confer differences in target-mediated as well as off-target adverse effects. Disease progression on covalent BTKis, driven by histologic transformation or selective expansion of BTK and PLCG2 mutated CLL clones, remains a major challenge in the field. Fixed duration combination regimens and reversible BTKis with non-covalent binding chemistry hold promise for the prevention and treatment of BTKi-resistant disease.

Identification of genes for variable regions of immunoglobulins that recognize sialylated glycans

We previously reported an antibody (clone ID: FR9, IgM-κ) that recognizes the sialyl oligosaccharide Neu5Acα2,6Galβ1,4GlcNAc as an epitope on glycoproteins and glycolipids. In the present study, we developed an antibody (clone ID: AFR45, IgM-κ) that recognizes Neu5Acα2,3Galβ1,4GlcNAc/Glc as an epitope on glycoproteins and glycolipids and compared the nucleotide and amino acid sequences of the immunoglobulin gene variable regions with those of FR9. The heavy chain variable (VH) regions of FR9 and AFR45 were encoded by different VH gene segments, each of which was composed of a characteristic D gene segment. The major differences between VH genes encoding various antibodies deposited in public databases and FR9 and AFR45 were identified in the D gene segment, indicating that D genes play a critical role in determining the epitope specificity of these antibodies. Surprisingly, although FR9 and AFR45 were obtained independently from different mice immunized with different immunogens, the light chain variable (VL) region nucleotide sequences were identical. The VL gene consisted of Igkv4-57 and Igkj4 gene segments (Igkv4-57j4), the sequences of which were identical to VL genes for a number of antibodies against meningococcal group C capsular polysaccharide deposited in public databases. As this polysaccharide is a sialic acid homopolymer, these results indicate that Igkv4-57j4 encodes a VL common to immunoglobulins that recognize sialylated glycans.

Biochemistry, structure, and cellular internalization of a four nanobody-bearing Fc dimer.

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

BCL-2 Inhibition as Treatment for Chronic Lymphocytic Leukemia.

At the end of the 1990s, with the advent of imatinib for chronic myeloid leukemia and rituximab for B cell lymphoproliferative diseases with CD20 expression, there was a great conceptual evolution in the treatment of onco-hematological diseases. Researchers from around the world and the pharmaceutical industry began to focus their efforts on the so-called target therapy used alone or associated with classic chemotherapeutic drugs. In chronic lymphocytic leukemia, the development of second-generation anti-CD20 antibodies, biosimilars, PI3K (phosphatidylinositol 3-kinases) inhibitors, BTK (Bruton's tyrosine kinase) inhibitors, and anti-bcl 2 drugs represented mainly by venetoclax brought new, broader, and more effective opportunities in the treatment of this disease. This breakthrough occurred mainly regarding patients with alteration in 17p or mutation of the p53 gene for whom selecting the new drugs that act on B cell signaling (BTK and PI3K inhibitors) in the first line is mandatory. In fit patients with immunoglobulin heavy chain mutation, it is still acceptable to use the chemotherapy regimen with fludarabine, cyclophosphamide, and rituximab (FCR) and, in those who do not fit or are not IgVH-mutated, bendamustine-rituximab regimen. However, the first-line use of ibrutinib or venetoclax associated with immunotherapy within the concepts of infinite (ibrutinib) or finite (venetoclax) treatment has been increasingly used. In the second line, venetoclax, ibrutinib, and idelalisib have become the preferred treatments. I believe that a process of instruction and decision shared with patients considering the risks-benefits-cost and access to treatments should guide the choices within these concepts. Another fundamental aspect to discuss is the objective of the treatment for chronic lymphocytic leukemia (CLL) for a specific patient: the increase progression-free survival and overall survival and/or the achievement of minimal residual disease. CLL is the most common leukemia in adults with a median age at diagnosis of 72 years. The clinical course is heterogeneous, and outcomes are influenced by individual clinical presentation and disease biology. Molecular and genomic factors, including fluorescence in situ hybridization (FISH) testing, karyotype, and immunoglobulin heavy chain variable region gene (IGHV) mutational status, are important to treatment decisions and to predict the clinical course. However, despite disease biology, the presence of active disease is the most important criteria to initiate treatment. In the past decade, target therapies that inhibit B cell receptor signaling pathways and, more recently, BCL2 antagonists have emerged as a new treatment paradigm: chemo-free with fixed duration therapy. Bruton's tyrosine kinase inhibitors (BTK) are a class of oral medications approved for frontline and relapsed disease, effective for achieving lasting response and disease control with a good safety profile. BTK inhibitors are an attractive option for high-risk patients who are not candidates for an intensive regimen. However, it is a continuous therapy, and drug resistance or severe adverse events could lead to treatment suspension. BCL2 antagonists are an attractive alternative to BTK inhibitors. Anti-apoptotic BCL2 is associated with tumor genesis and chemotherapy resistance. The BCl2, an anti-apoptotic protein located in the mitochondrial membrane, is a major contributor to the pathogenesis of lymphoid malignancies and is overexpressed in CLL cells promoting clonal cell survival. Venetoclax is a potent and selective member of the BH3 mimetic drugs and a physiologic antagonist of BCL2. Venetoclax has demonstrated quick and durable responses in naïve and relapsed or refractory CLL (r/r CLL) patients, including high-risk patients. Furthermore, it has shown deeper responses, achieving a higher incidence of negative minimal residual disease (MRD) with a fixed duration therapy. In the past decade, there was a remarkable progress in CLL treatment. However, neither of the new target therapies is considered curative or free of toxicity. This article will focus on the treatment approach of CLL patients with BCl2 antagonists. Treatment strategy (combined versus monotherapy; continuous versus limited duration therapy), toxicity profile, and future directions will be exposed in this review.

Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation.

Antigen-activated B cells diversify variable regions of B cell antigen receptors by somatic hypermutation in germinal centers (GCs). The positive selection of GC B cells that acquire high-affinity mutations enables antibody affinity maturation. In spite of considerable progress, the genomic states underlying this process remain to be elucidated. Single-cell RNA sequencing and topic modeling revealed increased expression of the oxidative phosphorylation (OXPHOS) module in GC B cells undergoing mitoses. Coupled analysis of somatic hypermutation in immunoglobulin heavy chain (Igh) variable gene regions showed that GC B cells acquiring higher-affinity mutations had further elevated expression of OXPHOS genes. Deletion of mitochondrial Cox10 in GC B cells resulted in reduced cell division and impaired positive selection. Correspondingly, augmentation of OXPHOS activity with oltipraz promoted affinity maturation. We propose that elevated OXPHOS activity promotes B cell clonal expansion and also positive selection by tuning cell division times.

An enhanced open sandwich immunoassay by molecular evolution for noncompetitive detection of Alternaria mycotoxin tenuazonic acid

Open sandwich enzyme-linked immunosorbent assay (OS-ELISA), a novel noncompetitive immunoassay format, has shown great potential in rapid detection for small molecules compared with traditional competitive format. Here, an enhanced OS-ELISA towards the mycotoxin tenuazonic acid (TeA) was developed for the first time based on heavy chain variable region (VH) and light chain variable region (VL) from the hybridoma cells (3F10) producing anti-TeA monoclonal antibody (mAb). The established OS-ELISA exhibited a limit of detection of 0.08 ng/mL, and was 13 times more sensitive than mAb-based indirect competitive ELISA (ic-ELISA). The proposed assay was also applied to detect TeA contents in juice, flour and tomato ketchup samples with satisfactory recoveries of 87.6%–111.3%. Finally, the great accuracy of the established OS-ELISA method was validated by the standard ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS).

Improving the yield of recalcitrant Nanobodies® by simple modifications to the standard protocol

Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (VHH) camelid IgGs. Their small size and single gene format make them amenable to various molecular biology applications that require a protein affinity-based approach. These features, in addition to their high solubility, allows their periplasmic expression, extraction and purification in E. coli systems with relative ease, using standardized protocols. However, some Nanobodies are recalcitrant to periplasmic expression, extraction and purification within E. coli systems. To improve their expression would require either a change in the expression host, vector or an increased scale of expression, all of which entail an increase in the complexity of their expression, and production cost. However, as shown here, specific changes in the existing standard E. coli culture protocol, aimed at reducing breakdown of selective antibiotic pressure, increasing the initial culture inoculum and improving transport to the periplasmic space, rescued the expression of several such refractory Nanobodies. The periplasmic extraction protocol was also changed to ensure efficient osmolysis, prevent both protein degradation and prevent downstream chelation of Ni2+ ions during IMAC purification. Adoption of this protocol will lead to an improvement of the expression of Nanobodies in general, and specifically, those that are recalcitrant.

Clinical Outcomes in Patients with Multi-Hit TP53 Chronic Lymphocytic Leukemia Treated with Ibrutinib.

TP53 aberration (TP53 mutation and/or 17p deletion) is the most important predictive marker in chronic lymphocytic leukemia (CLL). Although each TP53 aberration is considered an equal prognosticator, the prognostic value of carrying isolated (single-hit) or multiple (multi-hit) TP53 aberrations remains unclear, particularly in the context of targeted agents. We performed deep sequencing of TP53 using baseline samples collected from 51 TP53 aberrant patients treated with ibrutinib in a phase II study (NCT01500733). We identified TP53 mutations in 43 patients (84%) and del(17p) in 47 (92%); 9 and 42 patients carried single-hit and multi-hit TP53, respectively. The multi-hit TP53 subgroup was enriched with younger patients who had prior treatments and unmutated immunoglobulin heavy-chain variable region gene status. We observed significantly shorter overall survival, progression-free survival (PFS), and time-to-progression (TTP) in patients with multi-hit TP53 compared with those with single-hit TP53. Clinical outcomes were similar in patient subgroups stratified by 2 or >2 TP53 aberrations. In multivariable analyses, multi-hit TP53 CLL was independently associated with inferior PFS and TTP. In sensitivity analyses, excluding mutations below 1% VAF demonstrated similar outcome. Results were validated in an independent population-based cohort of 112 patients with CLL treated with ibrutinib. In this study, single-hit TP53 defines a distinct subgroup of patients with an excellent long-term response to single-agent ibrutinib, whereas multi-hit TP53 is independently associated with shorter PFS. These results warrant further investigations on prognostication and management of multi-hit TP53 CLL.See related commentary by Bomben et al., p. 4462.

Plasmodium falciparum-specific B cell responses in malaria-susceptible children and malaria-immune adults

Abstract Malaria is a vector-borne disease caused by parasites of the Plasmodium genus that infect millions of people annually, with P. falciparum being the most common and deadliest species. Development of immunological protection against clinical malaria requires chronic or repetitive parasite exposure, is mediated by IgG, and is correlated with antibodies against proteins expressed by merozoites, the free form of the parasite that invades erythrocytes. The goal of our study was to better understand the development of this protective antibody response, using merozoite surface protein 1 (MSP1) as a model antigen. MSP1 is highly abundant on the merozoite surface, highly immunogenic, and highly variable between parasite strains. Using B cell tetramers, we isolated MSP1-specific IgM+ and IgG+ memory B cells (MBCs) from immune adults and non-immune children living in Tororo, Uganda, a region of high transmission. In children, MSP1-specific B cells were mainly unswitched (IgM+) classical MBCs (cMBCs; CD21+ CD27+), while adults showed enrichment for class-switched (IgG+) cMBCs. Sequence analysis of the heavy chain variable regions revealed that MSP1-specific MBCs from adults carried higher rates of somatic hypermutation and showed extensive clonal expansion compared to those from children. When expressed as IgG, antibodies from IgM+ MBCs showed low avidity to MSP1, while those from IgG+ MBCs displayed strong reactivity with recombinant MSP1 and whole merozoites and inhibited parasite growth. These results suggest that extensive evolution of B cell responses takes place as a result of life-long P. falciparum exposure, which may be essential for the development of protection against malaria.