Panel Of Monoclonal Antibodies Research Articles

Key markers for diagnosis of minimal residual disease in multiple myeloma

Introduction. Therapeutic advances in recent years, the appearance on the market each time of new drugs that allow patients to achieve complete remission, a long period without progression dictate the need to control treatment by monitoring residual disease in multiple myeloma. Monitoring of multiple myeloma is the detection of a small number of tumor cells after therapy in a patient, which may be the cause of recurrence of the disease – control of minimal residual disease (MRD). This article discusses the key diagnostic markers (CD45, CD56 and CD19) of minimal residual multiple myeloma disease at initial diagnosis and after induction therapy. There are various diagnostic methods of research that can reliably assess the response to therapy and predict the occurrence of relapse. The main methods for detecting MRD are allele-specific polymerase chain reaction, next-generation sequencing and multicolor flow cytometry. The diagnosis of MRD by flow cytometry is the most widely used quite fast, quantitative, sensitive and affordable method, it is used for the primary diagnosis of multiple myeloma, as well as for monitoring MRD. It is important to identify the aberrant immunophenotype during the initial diagnosis for the correct subsequent assessment of MRD. MRD of multiple myeloma is considered an important component in the course of patient therapy before hematopoietic stem cell autotransplantation to assess the effectiveness of therapy, control and prognosis of the disease.The aim of the work MRD-study with the key diagnostic markers in multiple myeloma (CD45, CD56 and CD19).Materials and methods. The study was carried out in 59 patients with multiple myeloma. For all patients, in addition to standard methods of diagnosis and staging of the disease, bone marrow morphology (myelogram) and immunophenotype were examined. Eight-color flow cytometry with a panel of monoclonal antibodies for the diagnosis of plasma cell tumors Euro-Flow 2012 was used to diagnose MRD.Results. MRD was assessed by markers CD45, CD56 and CD19 in patients with multiple myeloma after induction therapy. The incidence of MRD-positivity for CD45 was 87.5 %, for CD56 – 97.5 %, for CD19 – 95.5 %. The incidence of MRD-negative status was for CD45 was 12.5 %, for CD56 – 2.5 % and for CD19 – 4,5 %.Conclusion. The use of a complex of these markers allows the most accurate determination of the MRD-negative status, taking into account the primary immunophenotype of malignant plasma cells.

177P Feasibility and usefulness of evaluation of immune status of lung cancer patients by EBUS/TBNA cells analysis

The efficacy of lung cancer immunotherapy with immune check inhibitors is well documented in clinical trials and in real life. However response to this therapy is individual and the biomarkers are continually investigated. The character of local tumour environment seems to be not without significance. We previously presented the usefulness of BAL in the evaluation of immune status of NSCLC patients. The aim of this study was to present the results of immune cells analysis in lymph nodes (LNs) aspirates obtained by EBUS/TBNA. 36 patients with primary lung cancer were investigated- an additional aspirate was taken for analysis by EBUS/TBNA during diagnostic bronchoscopy. The proportion of immune cells were determined in LNs aspirates and, for comparison, in peripheral blood (PB) by multiparameter flow cytometry. A panel of monoclonal antibodies was used for detection of: CD3, CD4, CD8 cells, NKT, NK, dendritic cells (DCs), regulatory Tcells (Tregs) and the expression of PD-1 and PD-L1 on lymphocytes and DCs. We found significant differences in the proportion of the main types of lymphocytes and dendritic cells between LNs aspirates and PB- the proportion of cells was higher in LNs than in PB and was highest in metastatic LNs (Table). Statistical analysis revealed important correlations between regulatory immune cells in metastatic lymph nodes. This may be evidence of consistent immune response in this local microenvironment. Significant differences (Mann Whitney test) in the proportion of immune cells in the blood (PB) and LNs.Table: 177PCellsPBLNsP<0.05CD317.2528.45*CD410.0516.10*CD86.859.95NKT0.100.80*NK2.353.35DCs0.040.71*PDL-1+ DCs1.6458.65*PD-1+ DCs1.8240.51*PD-1+ T cells10.4125.02*Dregs0.2222.50*Effector T cells2.405.80*Th1727.4534.15Tregs0.301.15 Open table in a new tab The results of this study confirmed the advantage of immune cells analysis in LNs aspirates obtained by EBUS/TBNA in the same time during lung cancer diagnosis.

The Age-Dependent Role of Th22, Tc22, and Tc17 Cells in the Severity of Pneumonia in COVID-19 Immunopathogenesis.

Coronavirus disease 2019 (COVID-19) has clinical manifestations ranging from mild symptoms to respiratory failure, septic shock, and multi-organ failure. Lymphocytes are divided into different subtypes based on their cytokine production pattern. In this study, we investigated the role of cytokine expressions of CD4+ T (T helper [Th]1, Th2, Th17, Th22) and CD8+ T cell subtypes (T cytotoxic [Tc]1, Tc2, Tc17, Tc22) in the pathogenesis of COVID-19. Peripheral blood mononuclear cells (PBMCs) were extracted with Ficoll by density gradient centrifugation from blood samples of 180 COVID-19 patients (children and adults) and 30 healthy controls. PBMCs were stimulated with PMA and Ionomycin and treated with Brefeldin A in the fourth hour, and a 10-colored monoclonal antibody panel was evaluated at the end of the sixth hour using flow cytometry. According to our findings, the numbers of Th22 (CD3+, CD4+, and interleukin [IL]-22+) and Tc22 (CD3+, CD8+, IL-22+) cells increased in adult patients regardless of the level of pneumonia (mild, severe, or symptom-free) as compared with healthy controls (p < 0.05). In addition, the number of Tc17 (CD3+, CD8+, and IL-17A+) cells increased in low pneumonia and severe pneumonia groups compared with the healthy controls (p < 0.05). Both IL-22 and IL-17A production decreased during a follow-up within 6 weeks of discharge. Our findings suggest that the increase in only IL-22 expressed Tc22 cells in the 0-12 age group with a general symptom-free course and higher levels of Th22 and Tc22 in uncomplicated adult cases may indicate the protective effect of IL-22. On the contrary, the association between the severity of pneumonia and the elevation of Tc17 cells in adults may reveal the damaging effect of IL-22 when it is co-expressed with IL-17.

Development of a new automated IL-6 immunoassay

ObjectivesQuantitative detection of interleukin-6 (IL-6) in serum and plasma can help monitor immune responses and the development of acute inflammation to guide patient management. We developed an IL-6 immunoassay for use with the automated ARCHITECT system for detecting an increase in the inflammatory response. MethodsImmunized mouse sera were tested and selected B-cells were harvested for fusion with myeloma cells. A panel of monoclonal antibodies were produced, from which capture and detection monoclonal antibodies for the prototype IL-6 immunoassay were selected and screened on the ARCHITECT instrument. The antibody pair that most effectively captured and detected IL-6 was selected to develop a prototype IL-6 immunoassay. Calibrator and panel preparations using an internal recombinant IL-6 standard were compared to serum panels prepared with the IL-6 International Standard 89/548. Assay specificity and spike recovery were determined, and assay sensitivity was compared with the Roche EUA Elecsys IL-6 assay run on the cobas analyzer. ResultsTwenty-one antibodies in 441 antibody pairs were screened. The prototype IL-6 assay showed high sensitivity with an estimated limit of detection of 0.317 pg/mL and limit of quantitation of <1.27. Spike recovery was 90%–110% in serum and plasma. The internal recombinant human IL-6 calibrator showed excellent stability for 63 days at 2–8 °C. The prototype IL-6 immunoassay was specific for IL-6, exhibited no cross reactivity to related cytokines and interleukins, and was 10-fold more sensitive than the Elecsys IL-6 assay. ConclusionsThe prototype ARCHITECT IL-6 automated immunoassay is a reliable and robust method for the quantitative determination of IL-6 in human serum and plasma.

Fusogenicity and neutralization sensitivity of the SARS-CoV-2 Delta sublineage AY.4.2

SummaryBackgroundSARS-CoV-2 lineages are continuously evolving. As of December 2021, the AY.4.2 Delta sub-lineage represented 20 % of sequenced strains in the UK and had been detected in dozens of countries. It has since then been supplanted by Omicron. The AY.4.2 spike displays three additional mutations (T95I, Y145H and A222V) in the N-terminal domain when compared to the original Delta variant (B.1.617.2) and remains poorly characterized.MethodsWe compared the Delta and the AY.4.2 spikes, by assessing their binding to antibodies and ACE2 and their fusogenicity. We studied the sensitivity of an authentic AY.4.2 viral isolate to neutralizing antibodies.FindingsThe AY.4.2 spike exhibited similar binding to all the antibodies and sera tested, and similar fusogenicity and binding to ACE2 than the ancestral Delta spike. The AY.4.2 virus was slightly less sensitive than Delta to neutralization by a panel of monoclonal antibodies; noticeably, the anti-RBD Imdevimab showed incomplete neutralization. Sensitivity of AY.4.2 to sera from vaccinated individuals was reduced by 1.3 to 3-fold, when compared to Delta.InterpretationOur results suggest that mutations in the NTD remotely impair the efficacy of anti-RBD antibodies. The spread of AY.4.2 was not due to major changes in spike fusogenicity or ACE2 binding, but more likely to a partially reduced neutralization sensitivity.FundingThe work was funded by Institut Pasteur, Fondation pour la Recherche Médicale, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, ANRS, the Vaccine Research Institute, Labex IBEID, ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR.

Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning.

Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen-specific antibodies at multiple distinct epitopes. This is a challenge, as stability enhancing mutations must be focused on the protein core, whereas successful computational stabilization algorithms typically select mutations at solvent-facing positions. In this study, we report the stabilization of SARS-CoV-2 Wuhan Hu-1 Spike receptor binding domain using a combination of deep mutational scanning and computational design, including the FuncLib algorithm. Our most successful design encodes I358F, Y365W, T430I, and I513L receptor binding domain mutations, maintains recognition by the receptor ACE2 and a panel of different anti-receptor binding domain monoclonal antibodies, is between 1 and 2°C more thermally stable than the original receptor binding domain using a thermal shift assay, and is less proteolytically sensitive to chymotrypsin and thermolysin than the original receptor binding domain. Our approach could be applied to the computational stabilization of a wide range of proteins without requiring detailed knowledge of active sites or binding epitopes. We envision that this strategy may be particularly powerful for cases when there are multiple or unknown binding sites.

Heterotypic interactions drive antibody synergy against a malaria vaccine candidate

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

COVID‐19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID‐19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID‐19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10‐color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39+ Tregs increased with disease severity. In contrast, CD39+ Tregs were decreased in juvenile patients in an age‐dependent manner. Overall, our study reveals an interesting profile of CD39‐expressing Tregs in adult and juvenile cases of COVID‐19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID‐19 cases.

Characterization and functional properties of a novel monoclonal antibody which identifies a B cell subpopulation in bursa of Fabricius

The bursa of Fabricius (BF) plays a central role in the development of B lymphocytes in birds. During embryonic development the BF primordium is colonized by myeloid and lymphoid prebursal stem cells to form the follicle buds, which ultimately develop into lymphoid follicles with a central medullary and an outer cortical region. Lympho-myeloid differentiation within the medulla is fundamental to normal B cell development. In contrast, the complexity of the cellular composition of the follicular cortex and its role in B cell differentiation has only recently begun to be studied. As an effort to characterize the different bursal cells we have produced a large panel of monoclonal antibodies (mAbs) by immunizing mice with a BF cell suspension of guinea fowl (Numida meleagris). One of these antibodies (clone: 7H3) was found to recognize a 80 kDa cell surface antigen expressed first in the yolk sac blood island of 2-day-old guinea fowl and chicken embryos, and later detected in the embryonic circulation and primary lymphoid organs. Double immunofluorescence revealed that chB6+ (Bu-1+) B cells of embryonic BF co-express the 7H3 antigen. 7H3 immunoreactivity of the bursal follicles gradually diminished after hatching and only a subpopulation of cortical B cells expressed the 7H3 antigen. In addition, in post-hatched birds 7H3 mAb recognizes all T lymphocytes of the thymus, peripheral lymphoid organs and blood. Embryonic BF injected with the 7H3 mAb showed a near complete block of lymphoid follicle formationIn conclusion, 7H3 mAb labels a new differentiation antigen specific for avian hematopoietic cells, which migrate through the embryonic mesenchyme, colonize the developing BF lymphoid follicles, and differentiate into a subpopulation of cortical B cells. The staining pattern of the 7H3 mAb and the correlation of expression with cell migration suggest that the antigen will serve as valuable immunological marker for studying the ontogeny of avian B cells.

SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses

SummaryOn 24th November 2021, the sequence of a new SARS-CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titers of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic Alpha, Beta, Gamma, or Delta are substantially reduced, or the sera failed to neutralize. Titers against Omicron are boosted by third vaccine doses and are high in both vaccinated individuals and those infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of the large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses and uses mutations that confer tight binding to ACE2 to unleash evolution driven by immune escape. This leads to a large number of mutations in the ACE2 binding site and rebalances receptor affinity to that of earlier pandemic viruses.

Исследование взаимосвязи состава микровезикул крови беременных с последующими плацента-ассоциированными осложнениями

Study Objective: To find the relationship between the levels of mitochondrial signal protein sequences in blood microvesicles of pregnant women and subsequent development of foetal growth retardation syndrome (FGRS), low birth-weight baby, preeclampsia (PE) or foetuses and newborns with normal weight and height. Study Design: Prospective comparative study. Materials and Methods. The microvesicular blood plasma fraction of women with placenta-associated complications in the outcome of previous pregnancies was studied as a risk group for their recurrence (n = 96). The study group was divided into 4 sub-groups: sub-group IА included 32 patients with pregnancies complicated with FGRS; sub-group IB comprised 20 patients with FGRS and PE; sub-group IC included 24 pregnant women, whose newborns were small for gestation age; sub-group ID was 20 pregnant women with a history of placenta-associated complications and abnormal Doppler velocimetry results, whose postnatal weight and height did not differ from normal values. The control group consisted of 20 women with a physiological course of pregnancy. The microvesicular fraction of the peripheral blood of the patients was obtained by differential plasma ultracentrifugation. Protein of the outer membrane of mitochondria (VDAC1), mitochondrial DNA transcription and replication activator (TFAM), recombinant protein of the outer membrane of mitochondria (MFN2), and TAZ protein encoding cardiolipin modelling in oxidative damage were measured by western blot analysis using a monoclonal antibody panel. Study Results. Plasma microvesicles in patients with subsequent placenta-associated complications demonstrated decreased levels of a number of mitochondrial proteins (VDAC1, TFAM, MFN2, OPA1). Levels of these proteins determine successful intercellular signaling on the adequate functional activity of mitochondria, their integrity and normal biogenesis. Conclusion. The obtained results allow to make a conclusion about a pronounced violation of mitochondrial biogenesis in the body of women with intrauterine growth restriction, which probably makes a serious contribution to the development of pathology and its progression. Keywords: foetal growth retardation syndrome, microvesicles, habitual abortion, placental insufficiency, preeclampsia, mitochondria, pregnancy.

ROR-1 Expression in the Diagnosis and Monitoring of Minimal Residual Disease in Chronic Lymphocytic Leukemia

Background. In view of similar morphological and phenotypic characteristics of some B-cell lymphoproliferative diseases and despite the known phenotype of tumor cells, a search is currently underway for new diagnostic markers, the expression of which remains stable during chronic lymphocytic leukemia (CLL) treatment and can be used for both diagnosis and assessment of residual tumor population. One of such markers is ROR-1. Aim. To assess the expression and feasibility of the ROR-1 marker using В-lymphocytes in minimal residual disease (MRD) dynamics and monitoring in CLL. Materials &amp; Methods. Hematological and immunophenotypic analyses were performed in 110 CLL patients (50 of them with newly diagnosed disease and 60 on therapy). In addition to that, 20 patients with reactive lymphocytosis and 32 donors were examined. The ROR-1 expression in В-lym-phocytes were measured with FACS Canto II flow cytometer using the following monoclonal antibody panel: CD45, CD19, CD20, and ROR-1. Results. The analysis showed that ROR-1 is essentially not expressed in normal and reactive В-lymphocytes and is detected in 100 % of CLL tumor cells both at disease onset and on therapy. The ROR-1 expression does not change during CLL treatment and can be used not only for CLL diagnosis but also for detection of MRD. Bone marrow aspirates (п = 64) and peripheral blood samples (п = 6) were analysed for MRD assessment by two methods: according to the standardized protocol, recommended by ERIC (European Research Initiative on CLL) in 2007, with FACS Canto II flow cytometer (BD Biosciences) and using DuraClone RE CLB Tube (Beckman Coulter) with Navious flow cytometer (Beckman Coulter).

Hepatitis B virus genotypes in patients with hepatitis D, determined by the panel of own development monoclonal antibodies

<h3>ВВЕДЕНИЕ</h3> Прямое определение генотипа вируса гепатита B (ВГВ) в образцах крови пациентов с гепатитом дельта может быть невозможно из-за недетектируемой концентрации ДНК ВГВ. Однако наличие достаточного количества поверхностного белка ВГВ (HBsAg) в них позволяет генотипировать ВГВ с помощью иммуноферментного анализа (ИФА) этого антигена, используя панель моноклональных антител (МАТ). <h3>ЦЕЛЬ РАБОТЫ</h3> Сопоставление результатов генотипирования ВГВ с помощью панели МАТ собственной разработки с результатами молекулярно-биологических исследований образцов пациентов с хроническим гепатитом B (ХГВ) и определение генотипов ВГВ в образцах сыворотки крови пациентов с гепатитом дельта. <h3>МАТЕРИАЛ И МЕТОДЫ</h3> Собраны 122 сыворотки крови пациентов с диагнозом ХГВ из Якутии и 211 сывороток крови от пациентов с гепатитом дельта из Якутии (12 образцов) и Тывы (199 образцов). Серотипы/генотипы ВГВ определяли методом ИФА с помощью разработанных реагентов. Молекулярно-биологические методы включали выделение ДНК ВГВ, амплификацию участка S-гена (713 нт), филогенетический анализ нуклеотидных последовательностей. <h3>РЕЗУЛЬТАТЫ</h3> С помощью набора МАТ в образцах пациентов ХГВ с детектируемой ДНК ВГВ (86 образцов) получено 95% (82/86) валидных результатов. Выявлены следующие генотипы: A — 32 (39%), C — 3 (4%), D — 47(57%). Для 81/82 (99%) образцов результаты определения генотипа ВГВ двумя методами совпали. В образцах пациентов с гепатитом дельта получено 96,2% валидных результатов при определении генотипа ВГВ с помощью МАТ (203/211) по сравнению с 3,8% стандартным молекулярно-биологическим методом (<i>p</i>&lt;0,001). Установлены следующие генотипы ВГВ: A — 17 (8,4%) образцов и D — 186 (91,6%). <h3>ВЫВОДЫ</h3> Разработанный тест с панелью МАТ позволяет достоверно определять генотип ВГВ и имеет преимущества по сравнению со стандартными молекулярно-биологическими методами при генотипировании ВГВ у пациентов с гепатитом дельта.

Highly Potent Immunotoxins Targeting the Membrane-distal N-lobe of GPC3 for Immunotherapy of Hepatocellular Carcinoma.

Glypican-3 (GPC3) has become a compelling target for immunotherapy of hepatocellular carcinoma, including antibody-drug conjugate (ADC), and ADC-like immunotoxin. To investigate the impact of epitopes on the potency of ADCs, current study generated a large panel of chicken monoclonal antibodies (mAbs) that targeted 12 different and over-lapping epitopes on GPC3. These mAbs demonstrated a very high affinity with Kd values in the range of 10-9-10-14 M, and the highest affinity (Kd value of 0.0214 pM) was 40-fold higher than the previously generated high-affinity mAb YP7 (Kd value of 0.876 nM). Additionally, these mAbs exhibited excellent thermostability with Tm values in the range of 45-82 °C. As a proof-of-concept study for ADC, we made immunotoxins (scFv fused with PE24, the 24-kDa cytotoxic domain of Pseudomonas exotoxin A) based on these mAbs, and we found that immunotoxins targeting the N-lobe of GPC3 were overall much more potent than those targeting the C-lobe and other locations. One representative N-lobe-targeting immunotoxin J80A-PE24 demonstrated 3 to 13-fold more potency than the hitherto best immunotoxin HN3-PE24 that was previously developed. J80A-PE24 could suppress tumor growth much greater than HN3-PE24 in a xenograft mouse model. Combination of J80A-PE24 with an angiogenesis inhibitor FGF401 showed additive effect, which dramatically shrank tumor growth. Our work demonstrated that, due to high affinity, excellent thermostability and potency, chicken mAbs targeting the N-lobe of GPC3 are appealing candidates to develop potent ADCs for immunotherapy of liver cancer.

Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2.

The B.1.1.529/Omicron variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings of our study. Here we found that B.1.1.529 is markedly resistant to neutralization by serum not only from patients who recovered from COVID-19, but also from individuals who were vaccinated with one of the four widely used COVID-19 vaccines. Even serum from individuals who were vaccinated and received a booster dose of mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies against all known epitope clusters on the spike protein, we noted that the activity of 17 out of the 19 antibodies tested were either abolished or impaired, including ones that are currently authorized or approved for use in patients. Moreover, we also identified four new spike mutations (S371L, N440K, G446S and Q493R) that confer greater antibody resistance on B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.

Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2

The B.1.1.529/Omicron variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings of our study. Here we found that B.1.1.529 is markedly resistant to neutralization by serum not only from patients who recovered from COVID-19, but also from individuals who were vaccinated with one of the four widely used COVID-19 vaccines. Even serum from individuals who were vaccinated and received a booster dose of mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies against all known epitope clusters on the spike protein, we noted that the activity of 17 out of the 19 antibodies tested were either abolished or impaired, including ones that are currently authorized or approved for use in patients. Moreover, we also identified four new spike mutations (S371L, N440K, G446S and Q493R) that confer greater antibody resistance on B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2. The B.1.1.529/Omicron variant of SARS-CoV-2 is resistant to neutralization by serum not only from patients who recovered from COVID-19, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines.

Non-denaturing affinity purification of soluble oligomeric Aβ from human brain using a novel calcium-sensitive monoclonal antibody.

The soluble fraction of human AD brain extracts contains the most bioactive oligomeric forms of Aβ (oAβ), which are low in abundance. Few methods exist to enrich for oAβ from soluble brain extracts without denaturing them. Purification of natural, human oAβ would allow further structural and biochemical study of this truly disease-relevant species. A panel of anti-oAβ monoclonal antibodies was developed by immunizing Trianni mice with synthetic Aβ aggregates. Clones were screened for oAβ selectivity and protection against human oAβ-induced injury in an in vitro neuritic integrity assay and in mouse hippocampal slice LTP recordings. Four antibodies with selectivity for oAβ over monomeric Aβ and protective effects were chosen for further study. Quantitative immunoprecipitations (IP followed by guanidine denaturation and monomer-specific ELISA) were performed on soluble extracts obtained by soaking cortical fragments bits in TBS to enrich for bioactive diffusible oAβ species. One antibody, B24, could IP oAβ only from non-dialyzed extracts. Iterative IPs were used to identify calcium as the soluble, dialyzable factor required for B24 binding to oAβ. Differential Scanning Fluorimetry (nano-DSF), Octet biolayer interferometry (BLI), and immunohistochemistry were further used to characterize B24 calcium-dependence and reversibility of binding to oAβ. B24 showed complete dependence on calcium at low millimolar levels for binding to soluble human oAβ, plaques, and synthetic Aβ protofibrils. Calcium induced a conformational change in B24. Binding of human soluble brain extracts to B24, washing, and elution with EGTA resulted in enrichment of oAβ. The recovered oAβ accounted for 20-40% of the initial input oAβ, accompanied by >400-fold reduction in total protein content. The recovered oAβ required guanidine hydrochloride for denaturation and detection by Aβ monomer-specific ELISA, implying that the recovered oAβ had not been denatured during purification. Calcium did not affect the size distribution of oAβ from human brain extracts by size exclusion chromatography. B24 is protective against natural oAβ-induced neuronal injury and binds oAβ in a calcium-dependent manner. This unique characteristic allows affinity purification of bioactive oAβ from human brain extracts for further study and drug development.

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Relationship between LDH and Mg in Monitoring of Hematological and Non-Hematological Malignant Diseases

Relationship between LDH and Mg in Monitoring of Hematological and Non-Hematological Malignant DiseaseAurelian Udristioiu,Titu Maiorescu University of Bucharest, Faculty of Medicine, Department of Molecular BiologyABSTRACTBackgroundMagnesium, which is the second most abundant into-cellular cation after potassium, plays a key role in regulating many cellular functions and enzymes, including ion channels, metabolic cycles, and signaling pathways.Aim of this study was to evaluate the correlation between the serum levels of lactate dehydrogenase (LDH) and magnesium (Mg) in patients diagnosed with hematological and non-hematological malignant diseases.MethodWe analyzed a cohort of patients (n = 75) comprising males (n = 36) and females (n =39) with a mean age of 57 years, who had cancer and were admitted to the oncology department. The biochemical parameters were measured running a Vitros 250 dry chemistry analyzer (Johnson & Johnson, USA) using the slides for multi-layer spectro-photometry measurements.These patients were closely monitored twice a week during treatment with specific cytostatic drugs and once a week during consolidation therapy, using specific tests for different types of cancer. In addition, with the cooperation of physicians and patients, they were performed along with hematological and biochemical tests, CBC with differential count and LDH and serum Mg levels, which can serve as markers for monitoring the progression of malignancies.Among the patients, 5 patients were diagnosed with lung cancer, 11 patients were diagnosed with breast cancer, 14 patients were diagnosed with colorectal cancer, 43 patients were diagnosed with chronic lymphocytic leukemia (CLL), 1 patient was diagnosed with acute promyelocytic leukemia (LAM-3) and 1 patient was diagnosed with chronic monocytic leukemia (CML).The CBC with the differential count, biochemistry samples, body radiography, ultrasound and computed tomography (CT) were used for the patient to establish the type of cancer diseases. An initial panel of monoclonal antibodies was used to determine the immune phenotypes of the subgroups of differentiated T cells and B cells. The diagnosis of LAM-3 was made based on blood smears, the examination of bone marrow (BM) aspirates, the evaluation of promyelocytes, (greater than 30% in BM), and the presence of a specific immune phenotype.ResultsThe results were interpreted for each patient based on medical history, clinical and para-clinical examinations and other signs of malignant diseases. Among the patients in this study, 55 patients (73%) exhibited normal serum levels of Mg (normal range value = 1.60-2.3 mg/dL; mean value = 2.2 mg/dL; SD = 0.2; p = 0.02) following cancer therapy. Six patients (8%) exhibited low levels of Mg (range = 0.60-1.50 mg/dL; mean value = 1.05 mg/dL). However, 12 patients (16%) displayed high levels of serum Mg (range =2.6-3.27 mg/dL; mean value = 2.89 mg/dL). The levels of serum lactic dehydrogenase (LDH) were also evaluated in patients newly diagnosed with cancer and in patients with unfavorable responses to the cancer therapy (range = 240-1330 U/L; mean value = 787 U/L; SD =1.33; p = 0.002; normal values 135-225 U/L), (Table 1).DiscussionsThe serum Mg level is increased via Mg²+ release from malignant tissues in patients with malignant disease prior to treatment with cytostatic drugs. In the different malignant diseases, the serum Mg values were high, normal or low, independent of the serum LDH values. The LDH levels remained elevated after initial cytostatic treatment until cancer remission.The number of chromosome copies in malignant tumors can be correlated with the total serum LDH values. LDH levels in cancer patients are elevated due to high levels of LDH-3 isoenzyme in patients with malignancies and high levels of LDH-4 and LDH-5 isoenzymes, elevated patients with cancer of liver, muscle, lung and tissue tissues. conjunctive. High concentrations of serum LDH damage the cell membrane. Thereafter, malignant cells become invasive and metastasizes.ConclusionsNormal levels of Mg with moderately increased LDH levels were observed in all patients who had cancer that was in the regression phase following good responses to a specific cancer therapy. Low levels of Mg with high levels of serum LDH were observed in all patients with poor prognosis and metastases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.