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2. 2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 – Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach)

Author

Pan, Luying, primary, Mora, Johanna, additional, Walravens, Karl, additional, Wagner, Leslie, additional, Hopper, Shirley, additional, Loo, Lina, additional, Bettoun, David, additional, Bond, Sarah, additional, Dessy, Francis, additional, Downing, Sean, additional, Garofolo, Fabio, additional, Gupta, Soumi, additional, Henderson, Neil, additional, Irwin, Chad, additional, Ishii-Watabe, Akiko, additional, Kar, Sumit, additional, Jawa, Vibha, additional, Joseph, Julie, additional, Malvaux, Ludovic, additional, Marshall, Jean-Claude, additional, McDevitt, Jessica, additional, Mohapatra, Susovan, additional, Seitzer, Jessica, additional, Smith, Justin, additional, Solstad, Therese, additional, Sugimoto, Hiroshi, additional, Tounekti, Omar, additional, Wu, Bonnie, additional, Wu, Yuling, additional, Xu, Yuanxin, additional, Xu, Joshua, additional, Yamamoto, Takenori, additional, Yang, Lin, additional, Torri, Albert, additional, Kirshner, Susan, additional, Maxfield, Kimberly, additional, Vasconcelos, Joao Pedras, additional, Abhari, Mohsen Rajabi, additional, Verthelyi, Daniela, additional, Brodsky, Eric, additional, Carrasco-Triguero, Montserrat, additional, Kamerud, John, additional, Andisik, Matthew, additional, Baltrukonis, Daniel, additional, Bivi, Nicoletta, additional, Cludts, Isabelle, additional, Coble, Kelly, additional, Gorovits, Boris, additional, Gunn, George R, additional, Gupta, Swati, additional, Millner, Anders Holm, additional, Joyce, Alison, additional, Kubiak, Robert J, additional, Kumar, Seema, additional, Liao, Karen, additional, Manangeeswaran, Mohanraj, additional, Partridge, Michael, additional, Pine, Samuel, additional, Poetzl, Johann, additional, Rajadhyaksha, Manoj, additional, Rasamoelisolo, Michele, additional, Richards, Susan, additional, Song, Yuan, additional, Swanson, Steven, additional, Thacker, Seth, additional, Wadhwa, Meenu, additional, Wolf, Andreas, additional, Zhang, Lucia, additional, and Zhou, Lin, additional

Published
2023
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4. Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate

Author

Lavoie, Brigitte D., Shaw, Gary S., Millner, Anders, and Chaconas, George

Subjects
Transposons -- Research, DNA binding proteins -- Physiological aspects, Escherichia coli -- Research, Molecular structure -- Analysis, Biological sciences
Abstract

The Escherichia coli HU flexer-Mu transpososome complex, present inside a higher-order transposition intermediate, contains a significant DNA bend. HU is a nonsequence-specific histone- and HMG-like DNA-binding protein. One HU heterodimer binds in a globally-symmetrical, site-specific and orientation-specific manner with Mu. Each HU heterodimer consists of two subunits that are functionally inequal. The HU-DNA complex shows major and minor groove cleavages.

Published
1996

5. CXC chemokine receptor 3 expression on CD34+hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon γ–inducible protein 10 and monokine induced by interferon γ

Author

Jinquan, Tan, Quan, Sha, Jacobi, Henrik H., Jing, Chen, Millner, Anders, Jensen, Bettina, Madsen, Hans O., Ryder, Lars P., Svejgaard, Arne, Malling, Hans-Jørgen, Skov, Per S., and Poulsen, Lars K.

Published
2000
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8. CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells

Author

Jinquan, Tan, Jacobi, Henrik H, Jing, Chen, Millner, Anders, Sten, Eva, Hviid, Lars, Anting, Liu, Ryder, Lars P, Glue, Christian, Skov, Per S, Jarman, Elizabeth, Lamberth, Kasper, Malling, Hans-Jørgen, Poulsen, Lars K, Jinquan, Tan, Jacobi, Henrik H, Jing, Chen, Millner, Anders, Sten, Eva, Hviid, Lars, Anting, Liu, Ryder, Lars P, Glue, Christian, Skov, Per S, Jarman, Elizabeth, Lamberth, Kasper, Malling, Hans-Jørgen, and Poulsen, Lars K

Abstract

Udgivelsesdato: 2003-Aug-15, We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.

Published
2003

9. Spontaneous and cytokine induced basophil adhesion evaluated by microtiter assay

Author

Quan, Sha, Poulsen, Lars K, Reimert, Claus Michael, Glue, Christian, Millner, Anders, Jensen, Bettina M, Jinquan, Tan, Stahl Skov, Per, Quan, Sha, Poulsen, Lars K, Reimert, Claus Michael, Glue, Christian, Millner, Anders, Jensen, Bettina M, Jinquan, Tan, and Stahl Skov, Per

Abstract

We have developed a microtiter assay for evaluating basophil spontaneous adhesion to extracellular matrix (ECM) proteins exemplified by fibronectin and cytokine induced basophil adhesion to bovine serum albumin (BSA). The percentage of basophils adhering to either ECM or BSA was quantified by the histamine content of the adhering basophils. The spontaneous adhesion to fibronectin was higher than to laminin and collagen type I. Both spontaneous adhesion to fibronectin and interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte/macrophage colony stimulating factor (GM-CSF) induced adhesion to BSA increased with time between 5 and 45 min. The histamine release in both spontaneous and induced basophil adhesion was lower than 3.1%. This microtiter assay is simple and reproducible and can be applied for basic and clinical studies using a limited number of partially purified basophils.

Published
2002

12. Cutting edge:Expression of the NF of activated T cells in eosinophils: Regulation by IL-4 and IL-5

Author

Jinquan, Tan, Quan, Sha, Jacobi, Henrik H., Reimert, Claus M., Millner, Anders, Hansen, Jens B., Thygesen, Charlotte, Ryder, Lars P., Madsen, Hans O., Malling, Hans Jørgen, Poulsen, Lars K., Jinquan, Tan, Quan, Sha, Jacobi, Henrik H., Reimert, Claus M., Millner, Anders, Hansen, Jens B., Thygesen, Charlotte, Ryder, Lars P., Madsen, Hans O., Malling, Hans Jørgen, and Poulsen, Lars K.

Abstract

We report that NF-AT1 and NF-AT4 are expressed cytoplasmically in resting eosinophils, whereas NF-AT2 and NF-AT3 have not been seen. Likewise, NF-AT1 mRNA and NF-AT4 mRNA have been detected in resting eosinophils, and their levels can be significantly up-regulated by the Th2-associated cytokines IL-4 and IL-5. There is no detectable NF-AT protein expression in the nuclei of resting eosinophils. However NFATs appear in the nuclei of IL- 4-, IL-5-, or ionomycin-stimulated eosinophils. Only NF-AT1 and NF-AT4, but not NF-AT2 and NF-AT3, have translocated into the nuclei in IL-4- or IL5- stimulated eosinophils. These findings delineate a novel pathway in the cytokine network in which Th2 lymphocytes 'control' eosinophils via the release of IL-4 and IL-5, and activation of NF-AT in eosinophils. The findings also suggest that a later feedback 'talking' may exist between eosinophils and Th2 lymphocytes.

Published
1999

13. Allergy Vaccine Engineering: Epitope Modulation of Recombinant Bet v 1 Reduces IgE Binding but Retains Protein Folding Pattern for Induction of Protective Blocking-Antibody Responses

Author

Holm, Jens, primary, Gajhede, Michael, additional, Ferreras, Mercedes, additional, Henriksen, Anette, additional, Ipsen, Henrik, additional, Larsen, Jørgen N., additional, Lund, Lise, additional, Jacobi, Henrik, additional, Millner, Anders, additional, Würtzen, Peter A., additional, and Spangfort, Michael D., additional

Published
2004
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14. CCR3 Expression Induced by IL-2 and IL-4 Functioning as a Death Receptor for B Cells

Author

Jinquan, Tan, primary, Jacobi, Henrik H., additional, Jing, Chen, additional, Millner, Anders, additional, Sten, Eva, additional, Hviid, Lars, additional, Anting, Liu, additional, Ryder, Lars P., additional, Glue, Christian, additional, Skov, Per S., additional, Jarman, Elizabeth, additional, Lamberth, Kasper, additional, Malling, Hans-Jørgen, additional, and Poulsen, Lars K., additional

Published
2003
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21. CXC chemokine receptor 3 expression on CD34+hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon ?–inducible protein 10 and monokine induced by interferon ?

Author

Jinquan, Tan, Quan, Sha, Jacobi, Henrik H., Jing, Chen, Millner, Anders, Jensen, Bettina, Madsen, Hans O., Ryder, Lars P., Svejgaard, Arne, Malling, Hans-Jørgen, Skov, Per S., and Poulsen, Lars K.

Abstract

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon ? (IFN-?)–inducible protein 10 (?IP-10) and monokine induced by IFN-? (Mig). We report the novel finding that CXCR3 is also expressed on CD34+ hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34+ progenitors. Freshly isolated CD34+progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase–polymerase chain reaction technique. ?IP-10 and Mig induced chemotaxis of GM-CSF–stimulated CD34+ progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block ?IP-10–induced and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colony-forming units—granulocyte-macrophage. ?IP-10 and Mig also induced GM-CSF–stimulated CD34+ progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of ?IP-10 and Mig but not of chemokine stromal cell–derived factor 1a. ?IP-10–induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF–stimulated CD34+progenitors. Moreover, ?IP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF–stimulated CD34+progenitors. These results indicate that CXCR3–?IP-10 and CXCR3–Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34+ hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34+ hematopoietic progenitors.

Published
2000
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22. Disruption of target DNA binding in Mu DNA transposition by alteration of position 99 in the Mu B protein11Edited by G. Smith

Author

Millner, Anders and Chaconas, George

Abstract

Target DNA binding by the Mu B protein is an important step in phage Mu transposition; however, the region of Mu B involved in target binding and the mechanism of the interaction are unknown. Previous studies have demonstrated that modification of Mu B with the sulfhydryl-specific reagent N-ethylmaleimide can selectively inhibit target DNA binding. We now show that individual mutation of the three cysteines in Mu B to serine results in proteins which are active in intermolecular strand transfer, but demonstrate variable levels of N-ethylmaleimide resistance. The data indicate that cysteine 99 is the primary site of modification affecting target DNA binding, with a minor contribution resulting from the derivitization of cysteine 129. These findings are confirmed by the construction of Mu B mutants containing a bulky side-chain at the individual cysteine to mimic the N-ethylmaleimide modified protein. The C99Y protein shows a complete loss in target-dependent strand transfer activity under standard reaction conditions and C129Y displays partial activity. The effect of the tyrosine substitutions is specific for target interaction as both mutants show wild-type activity in their ability to stimulate the Mu transposase to perform donor cleavage and intramolecular strand transfer. Finally, a target dissociation assay has shown that the C99Y-DNA complex generated in the presence of ATP-γ-S has a drastically reduced half-life as previously found for N-ethylmaleimide treated wild-type Mu B. Modification of cysteine 99 is proposed to block target DNA binding by causing steric interference near the DNA binding pocket.

Published
1998
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23. 2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness ( Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

Author

Loo L, Harris S, Milton M, Meena, Lembke W, Berisha F, Bertholet S, Dessy F, Dodge R, Fang X, Fiscella M, Garofolo F, Gorovits B, Gupta S, Jawa V, Ishii-Watabe A, Long B, Lu Y, Mack T, McGuire K, Nolan K, Pan L, Potthoff B, Purushothama S, Smith D, Solstad T, Sonderegger I, Taddeo F, Tangri S, Wagner L, Wu B, Xu Y, Kirshner S, Verthelyi D, Yan H, Maxfield K, Pedras-Vasconcelos J, Abhari MR, Gupta S, Wu Y, Rajadhyaksha M, Andisik M, Baltrukonis D, Cherry E, Cludts I, Gunn G, Millner AH, Jordan G, Kar S, Kubiak R, Lotz GP, Palmer R, Peng K, Poetzl J, Richards S, Savoie N, Staack RF, Stubenrauch K, Wadhwa M, Waxenecker G, Yang TY, and Zhang L

Subjects
Biomarkers analysis, CRISPR-Cas Systems, Cell- and Tissue-Based Therapy, Humans, Immunotherapy, Active, Polymerase Chain Reaction, Receptors, Chimeric Antigen, Vaccines
Abstract

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Published
2022
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24. Chemical modification of birch allergen extract leads to a reduction in allergenicity as well as immunogenicity.

Author

Würtzen PA, Lund L, Lund G, Holm J, Millner A, and Henmar H

Subjects
Adsorption, Allergens chemistry, Allergens immunology, Allergoids, Alum Compounds chemistry, Animals, Antigens, Plant chemistry, Antigens, Plant immunology, Cell Line, Glutaral chemistry, Histamine metabolism, Humans, Hypersensitivity, Immunization, Immunoglobulin G blood, Immunoglobulin G immunology, Mice, Mice, Inbred BALB C, Plant Extracts chemistry, Plant Extracts immunology, Plant Proteins chemistry, Plant Proteins immunology, T-Lymphocytes drug effects, T-Lymphocytes immunology, Allergens pharmacology, Antigens, Plant pharmacology, Betula immunology, Plant Extracts pharmacology, Plant Proteins pharmacology
Abstract

Background: In Europe, specific immunotherapy is currently conducted with vaccines containing allergen preparations based on intact extracts. In addition to this, chemically modified allergen extracts (allergoids) are used for specific allergy treatment. Reduced allergenicity and thereby reduced risk of side effects in combination with retained ability to activate T cells and induce protective allergen-specific antibody responses has been claimed for allergoids. In the current study, we compared intact allergen extracts and allergoids with respect to allergenicity and immunogenicity., Methods: The immunological response to birch allergen extract, alum-adsorbed extract, birch allergoid and alum-adsorbed allergoid was investigated in vitro in human basophil histamine release assay and by stimulation of human allergen-specific T cell lines. In vivo, Bet v 1-specific IgG titers in mice were determined after repetitive immunizations., Results: In all patients tested (n = 8), allergoid stimulations led to reduced histamine release compared to the intact allergen extract. However, the allergoid preparations were not recognized by Bet v 1-specific T cell lines (n = 7), which responded strongly to the intact allergen extract. Mouse immunizations showed a clearly reduced IgG induction by allergoids and a strongly potentiating effect of the alum adjuvant. Optimal IgG titers were obtained after 3 immunizations with intact allergen extracts, while 5 immunizations were needed to obtain maximal response to the allergoid., Conclusion: The reduced histamine release observed for allergoid preparations may be at the expense of immunological efficacy because the chemical modifications lead to a clear reduction in T cell activation and the ability to induce allergen-specific IgG antibody responses., (Copyright 2007 S. Karger AG, Basel.)

Published
2007
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