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.