Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fc␥ receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fc␥ receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.antibody-dependent cell-mediated cytotoxicity ͉ Fc␥R ͉ protein engineering ͉ cancer

Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals. We observed that prolonged exposure due to FcRn-mediated enhancement of half-life improved antitumor activity of Fcengineered antibodies in an hFcRn/Rag1 −/− mouse model. This bridges the demand for dosing convenience with the clinical necessity of maintaining efficacy.The well-established role of FcRn in IgG serum turnover has been the foundation for Fc engineering efforts aimed at improving the pharmacokinetic (PK) properties of antibodies 1, 2. Despite contrary results about the relationship between FcRn affinity and half-life3 , 4, a number of such PK engineering studies in non-human primates, whose FcRn is similar to that of human, have demonstrated increased half-life by engineered antibody variants [5][6][7][8] . Yet while the successful extension of half-life in PK experiments bodes well for the prospect of improving clinical dosing, a critical gap remains. For half-life extension technologies to be of practical use, efficacy of a biotherapeutic with longer half-life must be preserved at longer dosing intervals. Although the relationship between drug exposure and efficacy is well-established, this correlation has not thus far been established for antibodies Fc-engineered for longer halflife.Rational design methods coupled with high-throughput protein screening were used to engineer a series of Fc variants with greater affinity for human FcRn. Variants were constructed in the context of the humanized anti-VEGF IgG1 antibody bevacizumab 9 (Avastin®, Genentech/ Roche), which is currently approved for the treatment of colorectal, lung, breast, and renal cancers. A description of the construction, production, and binding studies of the antibodies is provided in the Supplementary Methods. Antibodies were screened for binding to human FcRn at pH 6.0 using Biacore. Engineered variants provide between 3 and 20-fold greater binding to FcRn at pH 6.0, with improvements due almost exclusively to slower off-rate (k off ) ( Supplementary Fig. 1, Supplementary Table 1). A lead variant M428L/N434S, subsequently selected principally based on its PK performance (see below), provided an 11-fold improvement in FcRn affinity at pH 6.0. This double substitution in the context of bevacizumab is referred to as Xtend™-VEGF. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA PK study was carried out in cynomolgus monkeys (macaca fascicularis) in order to evaluate the capacity of the variants to improve serum half-life in monkeys. A description of these experiments is provided in the Supplementary Methods. Binding improvements of the variants to monkey FcRn at pH 6.0 were comparable to improvements for human FcRn, and the rank order of the variants i...

Antibodies are the most rapidly growing drug class and have a major impact on human health, particularly in oncology, autoimmunity and chronic inflammatory diseases. Many of the best understood and most tractable cell surface and secreted targets with known roles in human diseases have been extensively exploited for antibody drug development. In this Review, we focus on emerging and novel mechanisms of action of antibodies and innovative targeting strategies that could extend their therapeutic applications, including antibody-drug conjugates, bispecific antibodies and antibody engineering to facilitate more effective delivery. These strategies could enable the pursuit of difficult to hit, less well-understood or previously undruggable targets - the 'high-hanging fruit'.

The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fc; receptors (Fc;R). Although an early goal has been enhanced Fc;RIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as Fc;RIIa. Here, we describe a set of engineered Fc variants with diverse Fc;R affinities, including a novel substitution G236A that provides selectively enhanced binding to Fc;RIIa relative to Fc;RIIb. Variants containing this substitution have up to 70-fold greater Fc;RIIa affinity and 15-fold improvement in Fc;RIIa/Fc;RIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual Fc;R to effector functions mediated by NK cells and macrophages. These experiments show that Fc;RIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor Fc;RIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.