Preclinical Development Humanization of anAnti-CCR4AntibodyThatKillsCutaneous T-Cell Lymphoma Cells and Abrogates Suppression by T-Regulatory Cells

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homingmalignant T cells that express high level of chemokine receptorCCR4,which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4þ CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitromechanistic studies showed that both complement-dependent cytotoxicity (CDC) and neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) likely mediated this effect. mAb1567 also exerts human NK cell–mediated ADCC activity in vitro. Moreover, mAb1567 also effectively inhibits chemotaxis of CD4þCD25high Tregs via CCL22 and abrogates Treg suppression activity in vitro. An affinity-optimized variant of humanized mAb1567, mAb2-3, was selected for further preclinical development basedon itshigherbindingaffinity andmorepotentADCCandCDCactivities.Taken together, thishigh-affinity humanized mAb2-3 with potent antitumor effect and a broad range of mechanisms of action may provide a novel immunotherapy for CTCL and other solid tumors. Mol Cancer Ther; 11(11); 2451–61. 2012 AACR.