--- _id: '8237' abstract: - lang: eng text: Monoclonal antibodies find broad application as therapy for various types of cancer by employing multiple mechanisms of action against tumors. Manipulating the Fc-mediated functions of antibodies that engage immune effector cells, such as NK cells, represents a strategy to influence effector cell activation and to enhance antibody potency and potentially efficacy. We developed a novel approach to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies. This entailed coupling single expression vector (pVitro1) antibody cloning, using polymerase incomplete primer extension (PIPE) polymerase chain reaction, together with simultaneous Fc region point mutagenesis and high yield transient expression in human mammalian cells. Employing this, we engineered wild type, low (N297Q, NQ), and high (S239D/I332E, DE) FcR-binding Fc mutant monoclonal antibody panels recognizing two cancer antigens, HER2/neu and chondroitin sulfate proteoglycan 4. Antibodies were generated with universal mutagenic primers applicable to any IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in small culture volumes, at high yields and within 12 days from design to purified material. Antibody variants conserved their Fab-mediated recognition of target antigens and their direct anti-proliferative effects against cancer cells. Fc mutations had a significant impact on antibody interactions with Fc receptors (FcRs) on human NK cells, and consequently on the potency of NK cell activation, quantified by immune complex-mediated calcium mobilization and by antibody-dependent cellular cytotoxicity (ADCC) of tumor cells. This strategy for manipulation and testing of Fc region engagement with cognate FcRs can facilitate the design of antibodies with defined effector functions and potentially enhanced efficacy against tumor cells. article_number: '1112' article_processing_charge: No article_type: original author: - first_name: Kristina M. full_name: Ilieva, Kristina M. last_name: Ilieva - first_name: Judit full_name: Fazekas-Singer, Judit id: 36432834-F248-11E8-B48F-1D18A9856A87 last_name: Fazekas-Singer orcid: 0000-0002-8777-3502 - first_name: Daniela Y. full_name: Achkova, Daniela Y. last_name: Achkova - first_name: Tihomir S. full_name: Dodev, Tihomir S. last_name: Dodev - first_name: Silvia full_name: Mele, Silvia last_name: Mele - first_name: Silvia full_name: Crescioli, Silvia last_name: Crescioli - first_name: Heather J. full_name: Bax, Heather J. last_name: Bax - first_name: Anthony full_name: Cheung, Anthony last_name: Cheung - first_name: Panagiotis full_name: Karagiannis, Panagiotis last_name: Karagiannis - first_name: Isabel full_name: Correa, Isabel last_name: Correa - first_name: Mariangela full_name: Figini, Mariangela last_name: Figini - first_name: Rebecca full_name: Marlow, Rebecca last_name: Marlow - first_name: Debra H. full_name: Josephs, Debra H. last_name: Josephs - first_name: Andrew J. full_name: Beavil, Andrew J. last_name: Beavil - first_name: John full_name: Maher, John last_name: Maher - first_name: James F. full_name: Spicer, James F. last_name: Spicer - first_name: Erika full_name: Jensen-Jarolim, Erika last_name: Jensen-Jarolim - first_name: Andrew N. full_name: Tutt, Andrew N. last_name: Tutt - first_name: Sophia N. full_name: Karagiannis, Sophia N. last_name: Karagiannis citation: ama: Ilieva KM, Singer J, Achkova DY, et al. Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields. Frontiers in Immunology. 2017;8. doi:10.3389/fimmu.2017.01112 apa: Ilieva, K. M., Singer, J., Achkova, D. Y., Dodev, T. S., Mele, S., Crescioli, S., … Karagiannis, S. N. (2017). Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields. Frontiers in Immunology. Frontiers. https://doi.org/10.3389/fimmu.2017.01112 chicago: Ilieva, Kristina M., Judit Singer, Daniela Y. Achkova, Tihomir S. Dodev, Silvia Mele, Silvia Crescioli, Heather J. Bax, et al. “Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields.” Frontiers in Immunology. Frontiers, 2017. https://doi.org/10.3389/fimmu.2017.01112. ieee: K. M. Ilieva et al., “Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields,” Frontiers in Immunology, vol. 8. Frontiers, 2017. ista: Ilieva KM, Singer J, Achkova DY, Dodev TS, Mele S, Crescioli S, Bax HJ, Cheung A, Karagiannis P, Correa I, Figini M, Marlow R, Josephs DH, Beavil AJ, Maher J, Spicer JF, Jensen-Jarolim E, Tutt AN, Karagiannis SN. 2017. Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields. Frontiers in Immunology. 8, 1112. mla: Ilieva, Kristina M., et al. “Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields.” Frontiers in Immunology, vol. 8, 1112, Frontiers, 2017, doi:10.3389/fimmu.2017.01112. short: K.M. Ilieva, J. Singer, D.Y. Achkova, T.S. Dodev, S. Mele, S. Crescioli, H.J. Bax, A. Cheung, P. Karagiannis, I. Correa, M. Figini, R. Marlow, D.H. Josephs, A.J. Beavil, J. Maher, J.F. Spicer, E. Jensen-Jarolim, A.N. Tutt, S.N. Karagiannis, Frontiers in Immunology 8 (2017). date_created: 2020-08-10T11:53:32Z date_published: 2017-09-11T00:00:00Z date_updated: 2021-01-12T08:17:39Z day: '11' doi: 10.3389/fimmu.2017.01112 extern: '1' intvolume: ' 8' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.3389/fimmu.2017.01112 month: '09' oa: 1 oa_version: Published Version publication: Frontiers in Immunology publication_identifier: issn: - 1664-3224 publication_status: published publisher: Frontiers quality_controlled: '1' status: public title: Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 8 year: '2017' ...