Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields
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Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields. / Ilieva, Kristina M; Fazekas-Singer, Judit; Achkova, Daniela Y; Dodev, Tihomir S; Mele, Silvia; Crescioli, Silvia; Bax, Heather J; Cheung, Anthony; Karagiannis, Panagiotis; Correa, Isabel; Figini, Mariangela; Marlow, Rebecca; Josephs, Debra H; Beavil, Andrew J; Maher, John; Spicer, James F; Jensen-Jarolim, Erika; Tutt, Andrew N; Karagiannis, Sophia N.
In: FRONT IMMUNOL, Vol. 8, 2017, p. 1112.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
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
Y1 - 2017
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
U2 - 10.3389/fimmu.2017.01112
DO - 10.3389/fimmu.2017.01112
M3 - SCORING: Journal article
C2 - 28959256
VL - 8
SP - 1112
JO - FRONT IMMUNOL
JF - FRONT IMMUNOL
SN - 1664-3224
ER -
