Enhanced NK cell activation via Fc receptor ligand cytokine combinations

Abstract Antibody dependent cellular cytotoxicity ADCC mediated by natural killer NK cells is a key effector function of therapeutic monoclonal antibodies mAb in use for treatment of established cancers mAbs communicate with NK cells via the low affinity Fc receptor IIIa Fc RIIIa mAbs bound to targets on cancer cells promote crosslinking of Fc RIIIa and activation of NK cells leading to both cytotoxicity and cytokine responses Polymorphisms in Fc RIIIa determine the avidity for mAb with the higher affinity variant correlating with more effective ADCC and better clinical outcomes As a result much of the development efforts of next generation therapeutic mAbs has centered on engineering mAbs with improved Fc RIIIa engagement Interleukin IL promotes the activation proliferation and cytotoxicity of NK cells Recombinant IL is approved for use in humans and has proven anti cancer effects but short half life and substantial toxicities have limited its use Fusion of IL to mAbs to produce `immunocytokinesandapos has several advantages over co administration of IL mAb such as reducing the amount of IL administered thereby reducing toxicities directing IL to NK cells that express Fc RIIIa the very subset of NK cells that initiates ADCC and localizing IL to the site of disease where NK cell activation may be most relevant to anti tumor responses We have developed novel modified Fc regions from human IgG consisting of open hinge tandem arrays of the HCH region `HCH polymersandapos that possess enhanced binding to low affinity Fc receptors Fc R In preliminary studies we developed anti Her Neu antibodies based on our HCH polymers and demonstrated their enhanced capacity to trigger ADCC against tumor cells We examined the role of exogenously added IL to potentiate ADCC from standard mAb Herceptin or from mAbs with enhanced Fc R binding We observed that mAbs with superior Fc R binding also had the greatest benefit from IL co administration Thus mAbs that have been optimized for NK cell activation via Fc R may be ideal candidates for further modification via fusion to IL One goal of the proposed studies is use our anti Her Neu mAbs that are already optimized for Fc R binding to create IL mAb fusions and to determine if the IL mAb fusions constitute a superior anti cancer therapeutic than the unmodified antibodies A second aspect of the proposed studies is to determine if non targeted IL optimized Fc fusions will function as an NK adjuvant for increasing ADCC responses when co administered with anti cancer mAbs In Aim studies we will produce IL fusions to targeted IL mAb and non targeted IL HSA forms of the HCH polymers In Aim we will determine the impact of IL fusion proteins on cytotoxicity phenotype and proliferative response of isolated NK cell subsets in vitro We will also determine whether the IL mAb fusions augment ADCC against Her expressing cell lines In Aim we will evaluate the antitumor effects of IL mAb and IL HSA fusions in vivo using SKOV xenografts in SCID mice and in an immunocompetent humanized immune system mouse model HSC NSG This combination of Aims is designed to deliver sufficient proof of principle for advancing the HCH polymer IL fusion proteins towards clinical development Narrative NK cells are the major innate lymphoid effector cells responsible for elimination of cancer cells One approach to improving NK cell mediated antibody dependent cellular cytotoxicity ADCC is to engineer antibodies that better engage Fc RIIIa the low affinity Fc receptor A second approach is to prime NK cells with IL so as to enhance NK cell mediated cytoxic responses including ADCC Since IL is highly toxic and has a short half life IL has been expressed as a fusion with antibodies to produce immunocytokines To date these approaches have been pursued separately In this proposal we aim to combine IL fusion with antibodies enhanced for Fc RIIIa engagement We hypothesize that by combining these two approaches we can produce anti cancer antibodies with improved therapeutic efficacy