Search

This article focuses on a list of forthcoming papers published in the journal. Some of them are: "Conformation and Domain Structure of the Non-Histone Chromosomal Proteins," "Differential Response of Cultured Parsley Cells to Elicitors from Two Non-Pathogenic Strains of Fungi," "Monoclonal Antibodies Against Platelet Membrane Glycoproteins," "Synthesis of Biotin-labelled Dexamethasone Derivatives: Novel Harmone-Affinity Probes," "On the Mechanisms of ATP-induced and Succinate-Induced Redistribution of Cations in Isolated Rat Liver Cells," "Shedding of Gangliosides From Calf Thymocytes."

Sakiyama Y, Kato R, Inoue S, Suzuki K, Itabe H, Yamamoto M. Detection of oxidized low-density lipoproteins in gingival crevicular fluid from dental patients. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01226.x. © 2009 John Wiley & Sons A/S Background and Objective: Oxidative modification of low-density lipoprotein (LDL) occurs in various diseased tissues and sites of local inflammation. For example, an increased plasma oxidized low-density lipoprotein (OxLDL) level is a well-known risk marker for cardiovascular diseases. Gingival crevicular fluid, the exudate from gingival tissues into the sulci, can be easily collected in a non-invasive manner. However, the possible presence of OxLDL in gingival crevicular fluid has not been studied. In this study, we established a procedure to measure OxLDL in human gingival crevicular fluid. Material and Methods: Human gingival crevicular fluid was sampled with paper points or paper strips. The gingival crevicular fluid samples from healthy gingival sulci (pocket depth < 4 mm, n = 14) were subjected to western blot and/or sandwich ELISA. The amounts of OxLDL and LDL were measured by sandwich ELISA using an anti-oxidized phosphatidylcholine monoclonal antibody and two anti-apolipoprotein B antibodies. Venous blood samples were analyzed biochemically. Results:  We tested two methods of gingival crevicular fluid collection, namely paper points and paper strips. Gingival crevicular fluid could be collected very safely with paper points and they showed good recovery of LDL and OxLDL throughout the analysis. Apolipoprotein B, the major protein component in LDL, was detected in gingival crevicular fluid by western blot, and OxLDL was found to be present in gingival crevicular fluid by ELISA. The OxLDL/LDL ratio in gingival crevicular fluid was 17.0 times higher than that in plasma. Conclusion: This is the first report to show the presence of apolipoprotein B and apolipoprotein B– oxidized phosphatidylcholine complex, which correspond to LDL and OxLDL, respectively, in gingival crevicular fluid. [ABSTRACT FROM AUTHOR]

An increased level of granulocyte‐macrophage colony‐stimulating factor has a potential role in the development of autoimmune diseases, and the neutralization of its activity by monoclonal antibodies is a promising therapy for some diseases. Here, the crystal structure of the Fab region of EV1007, a fully human antibody expressed in Chinese hamster ovary cells that was developed from human peripheral blood mononuclear cells, is described. The structure closely resembles that of MB007, which is the Fab region of the same antibody expressed in Escherichia coli [Blech et al. (2012), Biochem. J.447, 205–215], except at the hinge regions between the immunoglobulin domains and the H3 loop region. This paper presents evidence for the flexibility of the hinge and H3 loop regions of the antibody based on the comparison of two independently solved crystal structures. [ABSTRACT FROM AUTHOR]

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist‐based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti‐OX40 antibody (mAb035‐hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti‐OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen‐specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies. [ABSTRACT FROM AUTHOR]

Summary: The study of cellular and molecular immunology is almost completely dependent on monoclonal antibody technology. Despite the relatively long history of monoclonal antibodies, there is still huge potential for developing novel and transformative uses for this technology. In this issue of Immunology, we present two such papers, one focussing on anti‐complement (C5) antibodies, the other on anti‐CD6‐specific antibodies. Both manuscripts describe novel ways that monoclonal antibodies may be used for scientific and potentially for therapeutic benefit. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV‐1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion‐stabilized HIV‐1 envelope (Env) trimer, BG505 DS‐SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4‐binding site (CD4bs) of vulnerability. Two of the vaccine‐elicited CD4bs‐targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a‐hinge region and human IgG1‐constant region (G36×3‐IgG2a and R27×3‐IgG2a), neutralized 96% of a multiclade 208‐strain panel at geometric mean IC80s of 0.314 and 0.033 µg mL−1, respectively. Cryo‐EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV‐1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2‐apex‐targeting broadly neutralizing antibody, CAP256V2LS. The resultant human‐llama bispecific antibody CAP256L‐R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV‐1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn‐Fc mice similar to the parent CAP256V2LS. Vaccine‐elicited llama nanobodies, when combined with V2‐apex broadly neutralizing antibodies, may therefore be able to fulfill anti‐HIV‐1 therapeutic and prophylactic clinical goals. [ABSTRACT FROM AUTHOR]

This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete-event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision-making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E-factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium-sized companies is the integrated continuous strategy (alternating tangential flow filtration (ATF) perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed-batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision-making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:854-866, 2017 [ABSTRACT FROM AUTHOR]

T-colony formation can be induced in PHA-stimulated peripheral blood mononuclear cells (PBM) from man, but not in PHA-stimulated purified T cells, the latter requiring the presence of factors produced by PHA-stimulated PBM and termed T-colony promoting activity (TCPA). In this paper, we demonstrate that interleukin-2 (IL-2), the growth hormone of T lymphocytes, controls T-colony formation. We show that: (i) IL-2 activity and TCPA produced by PHA-activated PBM are co-purified by gel filtration and chromatography on blue agarose, a procedure which yields a 850-fold IL-2 purification; (ii) recombinant IL-2, produced by genetically manipulated Eseherichia coli, can induce T-colony formation in PHA-stimulated purified T cells; (iii) Monoclonal antibody against the IL-2 receptor (anti-Tac antibody) completely inhibits the T-colony formation in PHA-stimulated PBM when directly added to the culture system. [ABSTRACT FROM AUTHOR]

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (γ,κ) tumour cells with spleen cells from (BALB/c x C57Bl/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-ε-3.57, NP-ε-15,88, NP-ε-91,58 and NP-ε-95,31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated that NP-ε-15,88, NP-ε-91,58 and NP-ε-95.31 all possessed λ1 (or possibly λ3) light chains; and that NP-ε-3,57 possessed λ2 light chains; NP-&elipson;-95,31 also expressed the P3-X20 derived, MOPC-21 κ light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-ε-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-ε- aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells. [ABSTRACT FROM AUTHOR]

The titer of anti‐severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) neutralizing antibodies (NAbs) in the human body is an essential reference for evaluating the acquired protective immunity and resistance to SARS‐CoV‐2 infection. In this study, a fluorescence‐quenching lateral flow immunoassay (FQ‐LFIA) is established for quantitative detection of anti‐SARS‐CoV‐2 NAbs in the sera of individuals who are vaccinated or infected within 10 min. The ultrabright aggregation‐induced emission properties encapsulated in nanoparticles, AIE490NP, are applied in the established FQ‐LFIA with gold nanoparticles to achieve a fluorescence "turn‐on" competitive immunoassay. Under optimized conditions, the FQ‐LFIA quantitatively detected 103 positive and 50 negative human serum samples with a limit of detection (LoD) of 1.29 IU mL−1. A strong correlation is present with the conventional pseudovirus‐based virus neutralization test (R2 = 0.9796, P < 0.0001). In contrast, the traditional LFIA with a "turn‐off" mode can only achieve a LoD of 11.06 IU mL−1. The FQ‐LFIA showed excellent sensitivity to anti‐SARS‐CoV‐2 NAbs. The intra‐ and inter‐assay precisions of the established method are below 15%. The established FQ‐LFIA has promising potential as a rapid and quantitative method for detecting anti‐SARS‐CoV‐2 NAbs. FQ‐LFIA can also be used to detect various biomarkers. [ABSTRACT FROM AUTHOR]

We have previously reported a new receptor (NC-2) for natural cytotoxicity (NC) on murine leucocytes, identified by monoclonal antibody D9 (mAb D9). Pretreatment of mouse spleen cells with different concentrations of mAb D9 in vitro blocked NC against WEHI-164, whereas natural killing (NK) activity against YAC-1 was unaffected. This paper reports the immune surveillance against the growth of WEHI-164 tumour cells in mice by NC-2 Cells . The kinetics of in vivo reduction in NC activity were investigated by treating BALB/ c and (CBA × C57BL/6) F1 mice with a single injection of 40 μg of mAb D9 and monitoring splenic NC activity by Cr-release assay at intervals from 24 h to 3 weeks. Control mice were injected with OKT8 irrelevant antibody. Results showed a significant ( P < 0.05) reduction in splenic NC activity within 24 h which persisted for up to 1 week. Similar results were also obtained when (CBA × C57BL/6) F1 mice were employed ( P < 0.001). In vivo tumour studies were undertaken to investigate the role of NC-2 cells in surveillance against tumour growth and metastasis of the WEHI-164 fibrosarcoma. When syngeneic BALB/ c mice were injected with 40 μg of mAb D9 and then challenged with 5 × 10 WEHI-164 cells, results showed significantly increased growth rate of the transplanted WEHI-164 fibrosarcoma and tumour nodules in the lungs of animals, when compared to control mice with normal NC activity. Our data support an innate surveillance in metastasis and growth of WEHI-164 fibrosarcoma in mice. [ABSTRACT FROM AUTHOR]

Radioimmunotherapy uses monoclonal antibodies that are still labeled with only one radioactive atom. The aim of this paper is to assess, by means of MCNPX simulations, the doses delivered around and throughout a solid tumor when the radioactive atom linked to each antibody is replaced by a 5 nm diameter nanoparticle composed of numerous radionuclides. A new model for a spherical vascularized tumor has been developed in which the antibody distributions inside the tumor can be uniform or heterogeneous. It is also possible to simulate a central necrotic core inside the tumor where the concentration of radiolabeled antibodies is assumed to be zero. Dosimetry calculations have been performed for the beta-emitting radionuclide 90Y2O3. Preliminary results show that the irregularity of vasculature and the presence of a necrotic core have a noticeable influence on the deposited dose profiles. Moreover, with a total activity of 5 and 34 MBq for tumor radii of 0.5 and 1.0 cm, respectively, viable tumor cells can receive doses of up to 50 Gy, even if high nonuniformity of the total activity is observed in the tumor. These simulations still require accurate information about antibody characteristics and necrosis sizes but clearly confirm that the use of monoclonal antibodies conjugated to nanoparticles could lead to a considerable enhancement of treatment efficacy against cancer. [ABSTRACT FROM AUTHOR]

Antibodies have long been recognized for their diagnostic and therapeutic potential. The rapidly increasing number of monoclonal antibodies approved for immunotherapy has paved the way to an even greater demand for these molecules. In order to satisfy this growing demand and to increase the production capacity, alternative systems based on antibody production in transgenic organisms are being actively explored. In this paper, we focus on transgenic plants as a promising system for the scale-up and processing of plant-made pharmaceuticals. In particular, we point out the advantages and limitations induced by glycosylation of plant-made antibodies for human therapy. [ABSTRACT FROM AUTHOR]

Three semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) models, Simeoni, Jumbe, and Hybrid, were used for the efficacy translation of RC88 from preclinical to clinical. RC88 is a mesothelin‐targeting antibody–drug conjugate for malignant solid tumor. In the preclinical study, the relationship between PKs and PDs was determined using the xenograft mouse model derived from ovarian cancer and lung cancer cell lines. A secondary parameter representing the efficacy index of the drug, termed as tumor static concentration (TSC), was calculated using the three semimechanistic PK/PD models. A mechanism‐based target‐mediated drug disposition model was used to predict the human PKs. TSC from mice and predicted human PK were integrated to predict human efficacy dose. Results showed that 2 cell lines were sensitive to drugs, and the predicted efficacy dose was between 0.82 and 1.96 mg/kg q1w. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Phytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,χ) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vχ chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214. [ABSTRACT FROM AUTHOR]

Thymocytes from mice with experimental Trypanosoma cruzi infection respond poorly to Con-A stimulation. However, the proliferative capacity of these cells is not impaired, as demonstrated by the fact that at high doses, exogenous rIL-2 restores thymidine uptake. This finding could be explained either by insufficient IL-2 production or by the appearance of inhibitory factors during T. cruzi infection. This paper shows that in response to Con A, IL-2 production is decreased in the model. Furthermore, the whole profile of cytokine production is modified, with a striking increase in IL-10, IFN-γ, IL-4, IL-5 and IL-6 production. The results indicate that IL-10 plus IFN-γ are responsible for the decrease in the Con A-induced proliferation since a normal proliferative response as well as normal IL-2 production can be restored if both cytokines are neutralized by adding their monoclonal antibodies (MoAbs). Evidence is provided also for an enhanced non-specific cytotoxicity of thymic cells from infected mice that might involve IL-4, IL-5 and IL-6. This is the first study demonstrating an alteration of thymic cell function by T. cruzi infection which results from overstimulation of IL-10 and IFN-γ production. [ABSTRACT FROM AUTHOR]

The question of whether there is a preferential use of certain V genes in T cells entering an inflamed joint has hitherto been studied mainly using unfractionated cells from synovial fluid and tissue respectively, and no clear answer lo the question has yet been provided. Concomitantly. evidence has been provided that the use of V genes may differ considerably between CD4+ and CD8+ T cells, and consequently that detection of biased V-gene expression within an inflammatory lesion may require separate analysis of the two T-cell subsets. In this paper we have therefore studied T-cell receptor V-gene expression in rheumatoid arthritis by means of double stainings of synovial fluid and blood for available anti-TCR monoclonal antibodies and antibodies to CD4 and CD8, respectively. Double stainings were also performed with anti-TCR antibodies and antibodies to activation markers HLA-DR and IL-2R. A certain bias towards the preferential use of certain V genes was seen particularly in the synovial fluid samples within both the CD4+ and CD8+ T-cell populations, but no uniform pattern was evident among the 35 patients investigated. [ABSTRACT FROM AUTHOR]

Two different groups of monoclonal antibodies (MAbs) directed to different epitopes on 30-kDa region proteins of Mycobacterium tuberculosis were isolated; MAbs 5F9, 5D5 and 5D2 reacted with a single 33-kDa protein band, whereas MAb 3A8 reacted with a distinct 29/33-kDa doublet when analysed by immunoblotting.. The present paper describes the distribution of MAbs defined epitopes in the 29/33-kDa region proteins in well-characterized subcellular fractions: cytosol plasma membrane, cell wall as well as culture filtrate of M. tuberculosis. MAbs 5F9, 5D5 and 5D2 reactive epitopes were found in cytosol, whereas 3A8 epitope is distributed in all cellular compartments of the mycobacterium as well as in the culture tittrate. Localization of these epitopes by indirect immunoflourescence and immunogold-labelling demonstrated that only 3A8 epitope is present on the cell surface of the mycobacterium. Both immunoblotting and ELISA showed that only MAb 3A8, and not 5F9, 5D5 and 5D2, reacted with secreted BCG 85 antigen complex of Mycobacterrium bovis BCG. Furthermore, using an MAb 3A8-Crippled affinity column, we purified antigen 3A8 from the cytosol fraction of M. tuberculosis. All these MAbs reacted with antigen 3AK with varying degrees of intensity, thus suggesting that they are directed to a single protein. Absence of 5F9, 5D5 and 5D2 epitopes in the cell wall, culture filtrate and BCG-85 complex suggests that these epitopes might have been lost during the processing of the same 33-kDa protein on its way out from cytosol to the cell wall or when the protein is secreted out into the culture filtrate. Our results demonstrate. for the first time, direct evidence of the presence of a 30-kDa region protein not only in secreted antigens but also in the cell wall and on the cell surface of the mycobacterium. [ABSTRACT FROM AUTHOR]

This paper describes the production and characterization of a panel of 15 mouse monoclonal antibodies selected for putative activity against V-region related or allotypic determinants of a single IgG1λ paraprotein obtained from a patient with malignant lymphoplasmacytoid lymphoma. The specificity of each reagent for epitopes on the heavy (H) or light (L) chain or for conformational determinants (CD) of the immunogen was determined and the ability of one reagent to compete with another for these sites investigated. The fine specificity of the antibodies was assessed by screening on a large series of normal and paraprotein-containing sera. One monoclonal showed specificity for the Glm(f) allotype. The 14 other reagents identified a minimum of nine different epitopes in the V region of the immunogen, with four antibodies detecting private conformationally determined idiotypic specificities. Eight determinants were V-region markers also expressed on other paraproteins. A total of 30 out of 159 different paraproteins cross-reacted with one or more of the antibodies. Four of the shared epitopes were λ-chain associated, three were H-chain associated and one was a conformational determinant. Homologies of λ chain were identified more frequently among other paraproteins than those of H chain. The relationship between epitope expression and H-chain class of paraprotein was not random. The frequency of expression of cross-reactivities in association with IgG1 proteins was always exceeded by higher frequency of epitope expression in association with other classes of H-chain isotype. The potential therapeutic value of such panels of characterized monoclonal reagents is discussed. [ABSTRACT FROM AUTHOR]

Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a Tall antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed. [ABSTRACT FROM AUTHOR]

The present paper describes a new monoclonal antibody (K 1361) raised against hairy cell leukaemia cells. Antibody K861 recognizes a molecule of approximately 40,000 molecular weight on human B cells. It reacts with B lymphocytes in the peripheral blood, in primary lymphoid follicles, in the mantle zone of secondary follicles, in interfollicular areas and in splenic marginal zone areas. However, germinal centre lymphoid cells do not express the antigen recognized by antibody KB6I. The antibody shows limited reactivity outside the lymphoid system, i.e. polymorphs, tissue macrophages endothelial cells in the hepatic sinusoids. Antibody K861 discriminates between different types of B- cell malignancies, reacting with the neoplastic cells in hairy cell leukaemia, chronic lymphocytic leukaemia (of B-cell type), prolymphocytic leukaemia and centrocytic lymphoma, but not with acute lymphoblastic leukaemia, germinal centre-derived lymphomas (other than centrocytic), Burkitt's lymphoma and lymphoblastic lymphoma. Antibody KB61 may be of value in the study of B-cell subpopulations and in the differential diagnosis of B-cell neoplasms. [ABSTRACT FROM AUTHOR]

In this paper we present experiments that indicate that, in man, most T-celI subpopulations produce interleukin-2 (IL-2), but that the main cell subpopulation which produces it, both upon activation with phytohaemagglutinin or in autologous mixed lymphocyte cultures, is that of autologous rosette-forming (Tar) T4+ T cells. Conversely, the main IL-2-responding T-cell subpopulation is that composed of T cells depleted of Tar (T-Tar) that are T8+ IL-2 was also found to be more effectively produced by Tar cells that do not bind peanut agglutinin (PNA) than by those that do. The PNA T4+ Tar cells were also found to respond best to interleukin- I (IL-I). [ABSTRACT FROM AUTHOR]

The results presented in this paper demonstrate that a mouse IgM monoclonal antibody (T-80) recognizes an antigen on cells of the T-lymphocyte lineage of sheep. However, this antibody does not identify all T cells, as 10-20% of thymocytes and some peripheral-blood T cells are negative. T-80- thymocytes reside in the medulla. The majority of cortical thymocytes are T-80+ and classified as dull cells on the basis of antigen density per cell as measured by flow microfluorometry. In contrast, T-80+ cells in the periphery can be categorized into two populations, i.e., dull cells and bright cells. Suggestive evidence was obtained that bright T-80+ cells are fast recirculating T cells, whereas dull ceils are sessile or less easily mobilizable T cells in the periphery. In foetal environment, over 90% of thymocytes and approximately 5% of spleen cells are T-80+ at 54 days of gestation (gestation period = 150 days), which may indicate that T-cell emigration from the thymus commences well before mid-gestation in sheep. [ABSTRACT FROM AUTHOR]

The role of antibodies in the destruction of pathogenic cells is a basic concept in immunology. The advent of monoclonal antibodies (MCA) has renewed interest in cancer immunotherapy because of their specificity, and because they can be linked to radionuclides, drugs and toxins. MCA of defined specificity are continuously produced by permanent cell lines using the hybridoma method. These MCA are chemically homogeneous, react with constant avidity to single antigenic determinants, and can be isolated in quantities previously not obtainable by conventional methods. There are many problems associated with the therapeutic use of MCA. In this paper such problems and possible solutions are described, followed by a review of the literature on monoclonal antibody therapy.

This paper describes a monoclonal antibody (mAb), anti-pig CD45 (MAC323), that is directed against a polymorphic determinant. A monomorphic anti-pig CD45 mAb (K252.IE4) bound Strongly to leucocytes from both MAC323+ and MAC323- pigs, demonstrating the absence of the epitope rather than the CD45 molecule. The MAC323 determinant was present on all leucocytes in positive pigs, exhibiting different expression levels on subsets (monocytes > lymphocytes > polymorphs), but was absent on red blood cells. Pigs lacking this determinant were healthy and grew normally. Careful selection of male and female SLAb/b pigs produced families that were either positive or negative for this epitope. Interbreeding within these families identified the genetic segregation of this variation, which is consistent with the CD45323 epitope being inherited as a simple dominant autosomal gene. The lack of this determinant in certain lines of inbred pigs has been used to study the homing, lifespan and tissue distribution of donor unlabelled MAC323- leucocytes and their subsets (using single- and two-colour immunocytological techniques) in MAC323- recipients after either intravenous injection or exchange transfusion, These results, identifying trafficking areas and subsets in constitutive lymphoid and inflammatory tissues, obtained using this genetic marker, extend those obtained previously using fluorescein isothiocyanate (FITC)-labelled donor cells. [ABSTRACT FROM AUTHOR]

We have previously shown that chlorpromazine (CPZ) inhibits tumour necrosis factor (TNF) production and protects against endotoxic shock in mice. In this paper we investigated the effect of pretreatment with CPZ, 4 mg/kg i.p. 30 min before, compared with dexamethasone (DEX; 3 mg/ kg) on the induction of other endotoxin (lipopolysaccharide; LPS)-induced cytokines in the serum of mice, i.e. interleukin-lα (IL-lα), IL-6 and IL-b, and TNF. We also studied the effect of CPZ on serum and spleen-associated TNF. Both DEX and CPZ inhibited TNF production, whereas induction of IL-b and IL-6 was inhibited by DEX but not by CPZ. DEX did not affect IL-l0, while CPZ potentiated its induction. CPZ also inhibited spleen-associated TNF induction in LPS- treated mice, suggesting an effect on the synthesis of TNF. CPZ inhibited TNF induction by Gram-positive bacteria (heat-killed Staphylococcus epidermidis) and by anti-CD3 monoclonal antibodies. Intraperitoneal administration of CPZ also inhibited the induction of brain-associated TNF induced by intra-cerebroventricular injection of LPS. Therefore, CPZ is a more specific inhibitor of TNF production than DEX; in particular, CPZ increased the induction of 1L-l0, which is a `protective' cytokine known to inhibit LPS toxicity and TNF production. CPZ inhibited TNF production in vivo, irrespective of the TNF stimulus used to induce TNF. Finally, CPZ did not induce the `rebound' effect of DEX that, when given 24 hr before LPS, potentiates TNF production, but it did inhibit TNF production after 24 hr. [ABSTRACT FROM AUTHOR]

Eosinophils interact with extracellular matrix proteins and endothelial cells through adhesion proteins belonging to the β1 and β2 subfamilies of integrins. Extending previous observations, we found that tumour necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor stimulated generation of superoxide anion by eosinophils plated on fibronectin-coated surfaces. As studies with adherent neutrophils indicated that TNF might act as activating leucocyte integrins to deliver signals involved in activation of cell functions, we investigated the effects of monoclonal antibodies (mAb) directed against VLA-4 (CD49d/CD29), LFA-I (CD1 la/CDI8), CR3 (CDI lb/ CD 18) or the common β2 subunit (CD 18) on generation of eosinophil toxic oxygen molecules and spreading. We show that cross-linking of members of both the β and the β2 integrin subfamilies triggers eosinophil respiratory burst and spreading. Evidence for the selectivity of anti-integrin mAb effects is derived from thc findings that isotype-matched mAb of other specificities (anti-class I MHC Ag, anti-β2-microglobulin, anti-CD4) did not trigger eosinophil functions. The findings presented in this paper suggest that integrin-dependent, cosinophil adhesion in sites of allergic reaction may be accompanied by release of toxic oxygen molecules involved in tissue damage. [ABSTRACT FROM AUTHOR]

This paper describes a T-cell targeting strategy based on the use of an antigen-monoclonal antibody heteroconjugate. A rat anti-idiotypic monoclonal antibody specific for a murine B-cell lymphoma was conjugated to the purified protein derivative (PPD) of tuberculin. This construct selectively delivered up to 4.5 &times 104 molecules of PPD onto each tumour cell. Targeted PPD was internalized for endosomal processing and was presented in association with the I-A class II restriction element to PPD-reactive T-helper (Th) cells. Activated Th cells were demonstrated to proliferate and secrete significant levels of tumour necrosis factor (TNF). Such lymphokine secretion was observed at a PPD concentration as low as 1 ng/mI. Despite the secretion of TNF, the S-cell lymphoma was found to be resistant to autonomous Th-mediated cytotoxicity. Targeted Th cells did, however, activate tumourcidal macrophages that subsequently mediated significant tumour cytostasis. Based on this observation, it is proposed that the targeting system described may be exploited as the basis for a future immunotherapeutic strategy. [ABSTRACT FROM AUTHOR]

Summary: This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan‐Hu‐1 RBD of SARS‐CoV‐2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human‐like, highly homogenous N‐linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well‐characterized mAbs, suggesting that it is a Class 4 RBD‐binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS‐CoV‐2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety‐friendly alternative platform for generating mAbs to address the evolving SARS‐CoV‐2 crisis. [ABSTRACT FROM AUTHOR]

Antibodies against Rho GDP‐dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti‐RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement‐fixing properties of anti‐RhoGDI2 antibodies were studied using imaging flow cytometry. Anti‐RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co‐localized with C3 on ECs. Binding of human IgG1 monoclonal anti‐RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti‐RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury. [ABSTRACT FROM AUTHOR]

Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post‐exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody‐dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell‐based reporter gene assay (RGA) for the determination of the ADCC activity of anti‐RABV antibodies using CVS‐N2c‐293 cells, which stably express the glycoprotein (G) of RABV strain CVS‐N2c as target cells, and Jurkat cells, which stably express FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT‐luc/FcγRⅢa cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti‐RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti‐RABV antibodies. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2D) is a major public health concern and is characterized by sustained hyperglycemia due to insulin resistance and destruction of insulin‐producing β cells. One pathological hallmark of T2D is the toxic accumulation of human islet amyloid polypeptide (hIAPP) aggregates. Monomeric hIAPP is a hormone normally co‐secreted with insulin. However, increased levels of hIAPP in prediabetic and diabetic patients can lead to the formation of hIAPP protofibrils, which are toxic to β cells. Current therapies fail to address hIAPP aggregation and current screening modalities do not detect it. Using a stabilizing capping protein, monoclonal antibodies (mAbs) can be developed against a previously nonisolatable form of hIAPP protofibrils, which are protofibril specific and do not engage monomeric hIAPP. Shown here are two candidate mAbs that can detect hIAPP protofibrils in serum and hIAPP deposits in pancreatic islets in a mouse model of rapidly progressing T2D. Treatment of diabetic mice with the mAbs delays disease progression and dramatically increases overall survival. These results demonstrate the potential for using novel hIAPP protofibril‐specific mAbs as a diagnostic screening tool for early detection of T2D, as well as therapeutically to preserve β cell function and target one of the underlying pathological mechanisms of T2D. [ABSTRACT FROM AUTHOR]

A monoclonal antibody targeting programmed cell death‐1 (PD‐1) is one of the most promising treatments for human cancers. Clinical studies in humans demonstrated that the anti‐PD‐1 antibody provides a long‐lasting tumour response. Previously, we established an anti‐canine PD‐1 therapeutic antibody (ca‐4F12‐E6), and the pilot clinical study demonstrated that the antibody was effective in dogs with oral malignant melanoma (OMM). However, two OMM cases were still undergoing treatment when the pilot study was published. Here, we describe the long‐term follow‐up of those two cases. Although both cases showed long‐term survival with complete response (CR), the tumour response differed; the effect onset was slow in one case and a durable response was observed in the second case even after treatment discontinuation. Secondary malignant tumours occurred during treatment in both cases. This follow‐up study revealed that ca‐4F12‐E6 maintains CR in dogs for more than 1 year. In addition, the pattern of tumour response was unique compared to conventional therapy. These results indicate that new evaluation criteria for tumour response may be necessary for immunotherapy in veterinary medicine. Long‐term follow‐up is necessary regardless of the short‐term treatment responsiveness. [ABSTRACT FROM AUTHOR]

CD137 (4‐1BB, TNFRSF9), an inducible T‐cell costimulatory receptor, is expressed on activated T cells, activated NK cells, Treg cells, and several innate immune cells, including DCs, monocytes, neutrophils, mast cells, and eosinophils. In animal models and clinical trials, anti‐CD137 agonistic monoclonal antibodies have shown anti‐tumor potential, but balancing the efficacy and toxicity of anti‐CD137 agonistic monoclonal antibodies is a considerable hindrance for clinical applications. Here, we describe a novel fully human CD137 agonistic antibody (PE0116) generated from immunized harbor H2L2 human transgenic mice. PE0116 is a ligand blocker, which is also the case for Utomilumab (one of the leading CD137 agonistic drugs); PE0116 partially overlaps with Urelumab's recognized epitope. In vitro, PE0116 activates NF‐κB signaling, significantly promotes T‐cell proliferation, and increases cytokine secretion in the presence of cross‐linking. Importantly, PE0116 possesses robust anti‐tumor activity in the MC38 tumor model. In vivo, PE0116 exhibits a good safety profile and has typical pharmacokinetic characteristics of an IgG antibody in preclinical studies of non‐human primates. In summary, PE0116 is a promising anti‐CD137 antibody with a good safety profile in preclinical studies. [ABSTRACT FROM AUTHOR]

This paper describes how specific hybridoma clones can be rapidly selected using paramagnetic beads coated with the antigen used for immunization. Spleen cells from a mouse immunized with fragment D dimer (DD) from plasminolysed fibrin were first fused with X-63 mouse myeloma cells. Paramagnetic monodisperse beads (precoated with sheep anti-mouse antibodies) were then coated with S4, a monoclonal antibody to DD, and subsequently with DD. Mixing such beads with the fused cells allowed selective harvesting of cells with membrane-expressed anti-DD gammaglobulins using a magnetic particle concentrator. Within 24 h, the cells spontaneously detached from the beads and were plated out on 96-well plates. Supernatants from the clones obtained were tested by the ELISA technique. Antibodies specific for DD were produced by 40-79% of the tested clones. It is concluded that it is possible to use antigen-coated paramagnetic beads to select, prior to cloning, hybridomas that produce specific antibodies. Implementation of this technique has significantly reduced costs and time in our efforts to obtain hybridoma clones of interest. [ABSTRACT FROM AUTHOR]

The antigen‐antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol‐like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross‐reactivity with 33 compounds with the binding affinity (expressed by IC50) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) between the antibody and the 33 alternariol‐like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q2 values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r2 values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D‐QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition. [ABSTRACT FROM AUTHOR]

Background: Recent studies showed that a single injection of human monoclonal allergen‐specific IgG antibodies significantly reduced allergic symptoms in birch pollen‐allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen‐specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE‐mediated responses. Methods: A cDNA‐VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope‐specificity and affinity to Bet v 1. Furthermore, cross‐reactivity to Bet v 1‐homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen‐induced basophil activation were investigated. Results: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross‐react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha‐helix at the C‐terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1‐induced basophil activation. Conclusion: We identified high‐affinity Bet v 1‐specific nanobodies that recognize an important IgE epitope and reduce allergen‐induced basophil activation revealing the first proof that allergen‐specific nanobodies are useful tools for future treatment of pollen allergy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Transglutaminase type 2 (TG2) catalyses formation of ε‐(γ‐glutamyl)‐lysine bonds between proteins, including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlies tissue scarring and fibrosis. Many transglutaminase inhibitors exist and allowed for proof‐of‐concept studies in disease models, but their lack of specificity for the TG2 isoform, and/or poor pharmacokinetic/pharmacodynamic properties have limited their clinical application. We sought to develop a high affinity TG2‐specific antibody against extracellular TG2 activity, with characteristics suitable for therapeutic development. Experimental Approach: Individual human TG2 domains were used to immunize mice and generate hybridomas. Supernatants were screened for inhibition of recombinant human TG2 activity, with TG2 specificity determined by ELISA. Key Results: Thirteen TG2‐specific, hybridoma supernatants inhibited human transamidation activity. Each hybridoma was cloned and the antibody mapped to an epitope in the TG2 core domain, using phage display panning of a TG2 fragment library. Four distinct inhibitory epitopes were determined. The most effective antibodies (AB1, DC1, and BB7) bound to amino acids 313–327 (catalytic core), with an IC50 of approximately 6–7 nM. The antibodies inhibit TG2 in human cells and block ECM accumulation in a primary human proximal tubular epithelial cell model of fibrosis. Only 7 antibodies inhibited rat TG2, all with higher IC50 values. Conclusions and Implications: We identified a preferred inhibitory epitope in human TG2, developed antibodies with required characteristics for clinical development, and established that targeted inhibition of extracellular TG2 transamidation activity is sufficient to modify fibrotic remodelling. [ABSTRACT FROM AUTHOR]

The poor recovery of large biomolecules is a well-known issue in reversed-phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state-of-the-art reversed-phase wide-pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150 000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene-bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42 000 and 443 000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are one of the most rapidly growing drug classes used for the treatment of cancer, infectious and autoimmune diseases. Complement‐dependent cytotoxicity (CDC) is one of the effector functions for antibodies to deplete target cells. We report here an efficient chemoenzymatic synthesis of structurally well‐defined conjugates of a monoclonal antibody with a rhamnose‐ and an αGal trisaccharide‐cluster to recruit natural anti‐rhamnose and anti‐αGal antibodies, respectively, to enhance the CDC‐dependent targeted cell killing. The synthesis was achieved by using a modular antibody Fc‐glycan remodeling method that includes site‐specific chemoenzymatic Fc‐glycan functionalization and subsequent click conjugation of synthetic rhamnose‐ and αGal trisaccharide‐cluster to provide the respective homogeneous antibody conjugates. Cell‐based assays indicated that the antibody‐rhamnose cluster conjugates could mediate potent CDC activity for targeted cancer cell killing and showed much more potent efficacy than the antibody‐αGal trisaccharide cluster conjugates for CDC effects. [ABSTRACT FROM AUTHOR]

With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use. Herein, a novel Fab‐like antibody fragment generated via an in silico‐based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. This construct, designated as FabCH3, maintains the natural N‐terminus and C‐terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin‐specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)‐specific Fab Ranibizumab (in vivo). The high‐resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity‐determining regions of FabCH3, explaining its enhanced stability and affinity. Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab‐like antibody fragments with higher stability and antigen‐binding affinity that can be used as a distinct class of antibody therapeutics. [ABSTRACT FROM AUTHOR]

Three-dimensional structures were determined for two crystal forms (orthorhombic P212121 and monoclinic C2) of the Fab from the humanized version of a murine monoclonal antibody (AF2) that possesses binding and potent neutralizing activity against human interferon γ (IFN-γ). This humanized antibody (HuZAF; USAN name fontolizumab) is currently in phase II clinical trials for the treatment of Crohn's disease. HuZAF exhibits binding and IFN-γ neutralizing capacities that closely approximate those of the original antibody. It is shown that HuZAF, whose VH domain was designed using a best-sequence-fit approach, is closer structurally to its mouse precursor than is a version whose VH was constructed using a human sequence with lower homology to the original mouse sequence. This work thus offers direct structural evidence in support of the best-sequence-fit approach and adds to previous results of biological and biochemical evaluations of distinctly engineered antibodies that also favored the use of a best-sequence-fit strategy. A second crystal type appeared during attempts to crystallize the Fab-IFN-γ complex. The antibody-antigen complex that existed in solution dissociated in the crystallization mixture. A conformationally altered but unliganded HuZAF protein crystallized in a different space group (C2), with two Fab molecules in the asymmetric unit. In this crystal lattice, no space was available for accommodating the IFN-γ antigen. Thus, there are currently three slightly different structures of the HuZAF Fab. [ABSTRACT FROM AUTHOR]

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold‐promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations. SYNOPSIS: Effective treatment options for SARS‐CoV‐2 infections/COVID‐19 are still sparse. This study revealed highly potent therapeutic nanobodies/single‐domain (VHH) antibodies that neutralize SARS‐CoV‐2 and its emerging immune‐escape mutants. Alpaca immune libraries yielded 45 VHHs (of 22 sequence classes) that target two epitopes of the SARS‐CoV‐2 receptor‐binding domain (RBD) and block infection.The lead nanobody monomers are hyperthermostable, bind the RBD with low‐picomolar affinity and neutralize the virus at a concentration of 0.2–0.7 micrograms per liter (IC99+).Enhancement of the nanobodies' avidity by trimerization with the collagen XVIII NC1 domain yields neutralizers that block SARS‐CoV‐2 at concentrations as low as 40 nanograms per liter (IC99+).Clinical candidates include nanobody trimers, tandem fusions and monomers that bind the major SARS‐CoV‐2 immune‐escape mutants with high affinity and neutralize, e.g., the Beta/B.1.351 variant."Fold‐promoting" nanobodies assist de novo protein folding in the E. coli cytosol, as demonstrated with nanobody⋅RBD complexes. [ABSTRACT FROM AUTHOR]

The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 Å resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P21212, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 Å. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate β-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop, it is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity. [ABSTRACT FROM AUTHOR]

The amino-acid sequence of the Fe fragment of a mouse monoclonal IgG2a molecule is presented. With the exception of one deletion, this region possesses the same length as that of the human γl chain or that of the rabbit γ chain. Identities between the Fc fragments of 3 animal species (human, mouse and rabbit) average 50% and are markedly more pronounced for the CH2 domain than for the CH3 domain, observation which is in agreement with an independent evolution of each homology region. Strikingly, the degree of conservation of the VH region is of the same order of magnitude as that observed for the Fc fragment. This parallelism in evolution is discussed. [ABSTRACT FROM AUTHOR]

The serotype b antigens have been reported to be associated with lipopolysaccharide. Using murine monoclonal antibodies specific for either a serotype b antigen or the Actinobacillus actinomycetemcomitans species, the relationship of the two epitopes to lipopolysaccharide was determined. Both the species-specific and serotype b-specific monoclonal antibodies bound to whole cells, vesicles and conventionally isolated lipopolysaccharide and polysaccharide material derived from A. actinomycetemcomitans culture supernatants. Serotype b-specific monoclonal antibodies bound to the polysaccharide of acid-hydrolyzed lipopolysaccharide. Species-specific monoclonal antibodies bound to both the polysaccharide and the lipid A fraction of lipopolysaccharide after acid hydrolysis. Polymyxin b partially inhibited the binding of the species-specific monoclonal antibodies to lipopolysaceharide and had no effect on the binding of the serotype b-specific monoclonal antibodies to lipopolysaccharide. Lipopolysaccharide from whole bacteria and polysaccharide material isolated from culture supernatants were separated by gel filtration chromatography in deoxycholate into fractions that contained serotype b antigen, both serotype b and species-specific antigens, or species-specific antigen. SDS-polyacrylamide gel electrophoresis and Western blotting analysis of the fractions revealed that the serotype b antigen was on a high-molecular-weight polysaccharide material. The species-specific antigen was on a ladder of lower-molecular-weight polysaccharides identical to the blot pattern of hpopolysaccharide molecules separated by polyacrylamide gel electrophoresis and stained with silver stain. Chemical analysis of the polysaccharide containing serotype b antigen revealed 85% ribose, 11% glucose, and no lipid. Chemical content of the species-specific antigenic material revealed a composition typical of lipopolysaccharide. [ABSTRACT FROM AUTHOR]

This article focuses on the comparison of polyclonal and monoclonal antibodies for serotyping infectious pancreatic necrosis virus (IPNV) strains isolated in eastern Canada. IPNV, the aetiological agent of an important disease of salmonids, is a member of the Birnaviridae. Using polyclonal antibodies, three main serotypes have been distinguished according to their kinetics of neutralization in the presence of excess antibody by cross neutralization tests. All American strains are related to serotype I, while European isolates have been classified in serotypes II or III. Asian strains could belong to any of these three serotypes.

Copyright of Journal of Clinical Periodontology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)