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

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

doi:10.3389/fimmu.2017.01112

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.

Highlights

Monoclonal antibodies for cancer therapy engender a range of anti-tumor functions
We devised a cloning strategy that allows the manipulation of monoclonal antibody Fc regions in order to influence Fc receptors (FcRs) binding properties and antibody effector functions
Alongside wild type (WT) equivalents, we generated Fc-mutant variants designed either to have diminished or enhanced binding to FcRs on human immune effector cells, and we exemplified this strategy by engineering panels of antibody clones that recognize two cancer-associated antigens

Summary

Introduction

Monoclonal antibodies for cancer therapy engender a range of anti-tumor functions. These may be defined by Fab region recognition of target antigen epitopes and by engagement of the Fc region with cognate Fc receptors (FcR) on a variety of effector cell subsets. Agonistic antibodies specific for the TNFR family molecules have been reported to be significantly more efficacious if they have higher affinity for the inhibitory receptor FcγRIIB and lower affinity for activating FcRs [19, 20] This further highlights the crucial importance of assessing the contributions of the Fc region in different antibody immunotherapy approaches for cancer and the unmet need for novel Fc-engineered antibody variants to serve the purposes of specific strategies. Developing a strategy to manipulate antibody Fc regions can help to dissect the mechanisms of action of anti-cancer antibodies, allowing analysis of Fab-mediated functions in the presence or absence of Fc-mediated activities Such approaches could find a broad applicability in therapeutic antibody engineering and translation [26,27,28]

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