TY - JOUR A1 - Schlör, Anja A1 - Hirschberg, Stefan A1 - Ben Amor, Ghada A1 - Meister, Toni Luise A1 - Arora, Prerna A1 - Pöhlmann, Stefan A1 - Hoffmann, Markus A1 - Pfänder, Stephanie A1 - Eddin, Omar Kamal A1 - Kamhieh-Milz, Julian A1 - Hanack, Katja T1 - SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications JF - Frontiers in Immunology N2 - Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy. KW - camelid heavy-chain-only antibodies KW - single domain antibodies KW - nanobodies KW - SARS-CoV-2 KW - neutralization KW - Omicron Y1 - 2022 U6 - https://doi.org/10.3389/fimmu.2022.930975 SN - 1664-3224 SP - 1 EP - 14 PB - Frontiers Media SA CY - Lausanne, Schweiz ER - TY - JOUR A1 - Engel, Robert A1 - Micheel, Burkhard A1 - Hanack, Katja T1 - Three-dimensional cell culture approach for in vitro immunization and the production of monoclonal antibodies JF - Biomedical materials : materials for tissue engineering and regenerative medicine N2 - The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein. KW - monoclonal antibody KW - hybridoma technology KW - in vitro immunization KW - 3D KW - cell culture KW - Geltrex Y1 - 2022 U6 - https://doi.org/10.1088/1748-605X/ac7b00 SN - 1748-6041 SN - 1748-605X VL - 17 IS - 5 PB - Inst. of Physics CY - London ER - TY - JOUR A1 - Czarnecka, Malgorzata A1 - Weichelt, Ulrike A1 - Rödiger, Stefan A1 - Hanack, Katja T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA JF - International Journal of Molecular Sciences N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures. KW - antibody KW - camelid antibody KW - heavy-chain-only antibody KW - miRNA KW - nucleic acids KW - novel biomarkers Y1 - 2022 U6 - https://doi.org/10.3390/ijms23116275 SN - 1422-0067 VL - 23 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz ET - 11 ER - TY - GEN A1 - Czarnecka, Malgorzata A1 - Weichelt, Ulrike A1 - Rödiger, Stefan A1 - Hanack, Katja T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1274 KW - antibody KW - camelid antibody KW - heavy-chain-only antibody KW - miRNA KW - nucleic acids KW - novel biomarkers Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-569142 SN - 1866-8372 SP - 1 EP - 18 ER - TY - GEN A1 - Schlör, Anja A1 - Hirschberg, Stefan A1 - Ben Amor, Ghada A1 - Meister, Toni Luise A1 - Arora, Prerna A1 - Pöhlmann, Stefan A1 - Hoffmann, Markus A1 - Pfänder, Stephanie A1 - Eddin, Omar Kamal A1 - Kamhieh-Milz, Julian A1 - Hanack, Katja T1 - SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1280 KW - camelid heavy-chain-only antibodies KW - single domain antibodies KW - nanobodies KW - SARS-CoV-2 KW - neutralization KW - Omicron Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-570124 SN - 1866-8372 IS - 1280 ER - TY - GEN A1 - Göthel, Markus A1 - Listek, Martin A1 - Messerschmidt, Katrin A1 - Schlör, Anja A1 - Hönow, Anja A1 - Hanack, Katja T1 - A New Workflow to Generate Monoclonal Antibodies against Microorganisms T2 - Mathematisch-Naturwissenschaftliche Reihe N2 - Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1174 KW - monoclonal antibody KW - antibody producing cell selection KW - hybridoma KW - epitope prediction Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-523341 SN - 1866-8372 IS - 20 ER - TY - JOUR A1 - Göthel, Markus A1 - Listek, Martin A1 - Messerschmidt, Katrin A1 - Schlör, Anja A1 - Hönow, Anja A1 - Hanack, Katja T1 - A New Workflow to Generate Monoclonal Antibodies against Microorganisms JF - Applied Sciences N2 - Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms. KW - monoclonal antibody KW - antibody producing cell selection KW - hybridoma KW - epitope prediction Y1 - 2021 U6 - https://doi.org/10.3390/app11209359 SN - 1454-5101 VL - 11 IS - 20 PB - MDPI CY - Basel ER - TY - JOUR A1 - Michelchen, Sophia A1 - Micheel, Burkhard A1 - Hanack, Katja T1 - In vitro immunization approach to generate specific murine monoclonal IgG antibodies JF - Journal of immunological methods : JIM N2 - Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophila in vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species. KW - Monoclonal antibody KW - Hybridoma technology KW - In vitro immunization KW - B cell activation Y1 - 2021 U6 - https://doi.org/10.1016/j.jim.2021.113149 SN - 0022-1759 SN - 1872-7905 VL - 499 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Fudickar, Werner A1 - Roder, Phillip A1 - Listek, Martin A1 - Hanack, Katja A1 - Linker, Torsten T1 - Pyridinium alkynylanthracenes as sensitizers for photodynamic therapy JF - Photochemistry and photobiology N2 - Photodynamic therapy (PDT) is a mild but effective method to treat certain types of cancer upon irradiation with visible light. Here, three isomeric methylpyridinium alkynylanthracenes 1op were evaluated as sensitizers for PDT. Upon irradiation with blue or green light, all three compounds show the ability to initiate strand breaks of plasmid DNA. The mayor species responsible for cleavage is singlet oxygen (O-1(2)) as confirmed by scavenging reagents. Only isomers 1m and 1p can be incorporated into HeLa cells, whereas isomer 1o cannot permeate through the membrane. While isomer 1m targets the cell nucleus, isomer 1p assembles in the cellular cytoplasm and impacts the cellular integrity. This is in accordance with a moderate toxicity of 1p in the dark, whereas 1m exhibits no dark toxicity. Both isomers are suitable as PDT reagents, with a CC50 of 3 mu m and 75 nm, for 1p and 1m, respectively. Thus, derivative 1m, which can be easily synthesized, becomes an interesting candidate for cancer therapy. Y1 - 2021 U6 - https://doi.org/10.1111/php.13554 SN - 0031-8655 SN - 1751-1097 VL - 98 IS - 1 SP - 193 EP - 201 PB - Wiley CY - Hoboken ER - TY - GEN A1 - Listek, Martin A1 - Hönow, Anja A1 - Gossen, Manfred A1 - Hanack, Katja T1 - A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 865 KW - Antibody generation KW - Assay systems Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-459893 SN - 1866-8372 IS - 865 ER -