Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

Kristina M. Ilieva; Judit Fazekas-Singer; Daniela Y. Achkova; Tihomir S. Dodev; Silvia Mele; Silvia Crescioli; Heather J Bax; Anthony Cheung; Panagiotis Karagiannis; Isabel Correa; Mariangela Figini; Rebecca Marlow; Debra H Josephs; Andrew J Beavil; John Maher; James F Spicer; Erika Jensen-Jarolim; Andrew N Tutt; Sophia N Karagiannis

doi:10.3389/fimmu.2017.01112

Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

Kristina M. Ilieva, Judit Fazekas-Singer, Daniela Y. Achkova, Tihomir S. Dodev, Silvia Mele, Silvia Crescioli, Heather J Bax, Anthony Cheung, Panagiotis Karagiannis, Isabel Correa, Mariangela Figini, Rebecca Marlow, Debra H Josephs, Andrew J Beavil, John Maher, James F Spicer, Erika Jensen-Jarolim, Andrew N Tutt, Sophia N Karagiannis

St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, King's College London, Guy's Hospital, London, United Kingdom.
University of Veterinary Medicine Vienna
School of Cancer Sciences, King's College London, Bermondsey Wing, Guy's Hospital, London, United Kingdom.
Fondazione IRCCS Istituto Nazionale dei Tumori (INT)
Breast Cancer Now Research Unit, School of Cancer & Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, United Kingdom.
King's College London, School of Biomedical Sciences, Department of Biochemistry & Randall Division of Cell and Molecular Biophysics, New Hunt's House, London, United Kingdom ; Department of Pharmacy, University of Naples "Federico II", Napoli, Italy.

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Abstract

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.

Original language	English
Article number	1112
Journal	Frontiers in Immunology
Volume	8
Issue number	SEP
Early online date	11 Sept 2017
DOIs	https://doi.org/10.3389/fimmu.2017.01112
Publication status	Published - 11 Sept 2017

Keywords

Journal Article

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Functionally Active Fc Mutant_ILIEVA_Accepted24Aug2017_Publishedonline11Sep2017_GOLD VoR (CC BY)Final published version, 7.94 MBLicence: CC BY

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Ilieva, K. M., Fazekas-Singer, J., Achkova, D. Y., Dodev, T. S., Mele, S., Crescioli, S., Bax, H. J., Cheung, A., Karagiannis, P., Correa, I., Figini, M., Marlow, R., Josephs, D. H., Beavil, A. J., Maher, J., Spicer, J. F., Jensen-Jarolim, E., Tutt, A. N., & Karagiannis, S. N. (2017). Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields. Frontiers in Immunology, 8(SEP), Article 1112. https://doi.org/10.3389/fimmu.2017.01112

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abstract = "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.",

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author = "Ilieva, {Kristina M.} and Judit Fazekas-Singer and Achkova, {Daniela Y.} and Dodev, {Tihomir S.} and Silvia Mele and Silvia Crescioli and Bax, {Heather J} and Anthony Cheung and Panagiotis Karagiannis and Isabel Correa and Mariangela Figini and Rebecca Marlow and Josephs, {Debra H} and Beavil, {Andrew J} and John Maher and Spicer, {James F} and Erika Jensen-Jarolim and Tutt, {Andrew N} and Karagiannis, {Sophia N}",

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month = sep,

day = "11",

doi = "10.3389/fimmu.2017.01112",

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Ilieva, KM, Fazekas-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, vol. 8, no. SEP, 1112. https://doi.org/10.3389/fimmu.2017.01112

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T1 - Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

AU - Ilieva, Kristina M.

AU - Fazekas-Singer, Judit

AU - Achkova, Daniela Y.

AU - Dodev, Tihomir S.

AU - Mele, Silvia

AU - Crescioli, Silvia

AU - Bax, Heather J

AU - Cheung, Anthony

AU - Karagiannis, Panagiotis

AU - Correa, Isabel

AU - Figini, Mariangela

AU - Marlow, Rebecca

AU - Josephs, Debra H

AU - Beavil, Andrew J

AU - Maher, John

AU - Spicer, James F

AU - Jensen-Jarolim, Erika

AU - Tutt, Andrew N

AU - Karagiannis, Sophia N

PY - 2017/9/11

Y1 - 2017/9/11

N2 - 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.

AB - 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.

KW - Journal Article

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DO - 10.3389/fimmu.2017.01112

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SN - 1664-3224

VL - 8

JO - Frontiers in Immunology

JF - Frontiers in Immunology

IS - SEP

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Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

Abstract

Keywords

UN SDGs

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