Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

Abstract

BackgroundCC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis.MethodologyUsing phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.SignificanceFor the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VHCDR3 targeting the Class A GPCR formyl-peptide receptor 1

“…A recent publication also reports the use of cell-based phage display selection to optimize scFv targeting CCR4, although the details are not described. 18 Partially purified Figure 3. Determination of the apparent affinity of Fpro0165 for human FPR1 and cynomolgus FPR1 using Schild analysis of activity in a CD11b upregulation assay using in primary human and cynomolgus neutrophils.…”

“…Similarly, in the recently published example of CCR4-expressing cell-based selection of a na€ ıve phage display scFv library, 1.1% of the scFv antibodies screened were specific for CCR4, although only 4 out of these 132 antibodies were unique. 18 For the in vitro affinity maturation of antibodies, however, non-specific background due to the use of cells is likely to be less of an issue since the starting point is an antibody already recognizing the protein of interest.…”

“…[15][16][17] A recent publication describes the use of intact cells for the isolation of anti-chemokine receptor 4 (CCR4) antibodies from a na€ ıve phage display human scFv library. 18 Therapeutic antibodies are often required to be of higher affinity than is typical for antibodies derived by immunization of animals or selected from na€ ıve in vitro display libraries; however, in vitro affinity maturation of an antibody is ideally carried out using a purified source of the target protein that is structurally and functionally relevant to ensure antibodies with the desired activity are obtained. 19,20 Therefore, affinity optimization of antibodies targeting complex integral membrane proteins such as GPCRs, particularly those with relatively small extracellular regions, is a significant challenge limiting the availability of suitable antibodies to modulate this important group of potential therapeutic targets.…”

A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer

“…In preclinical experiments we found that Affi-5, a fully human anti-CCR4 antibody with antagonistic activity (described in ref. 19), has antitumor activity in a renal cancer model. Inhibition of CCR4 did not reduce the proportion of CCR4-positive infiltrating leukocytes in the tumor microenvironment but altered the phenotype of the immune infiltrate, affecting in particular the phenotype of myeloid cells and increasing the number of infiltrating NK cells.…”

DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops

“…2 While modulating antibodies against Class B and C GPCRs, and against transporters and ion channels (e.g., P2£3 and P2£7) with relatively large extracellular domains (ECD), have been reported, [3][4][5] functional antibodies against class A GPCRs and most of the transporters and ion channels with a small extracellular domain and small extracellular loops have been more challenging, and examples of mAbs raised against these targets are rare. 6,7 Hybridoma technology and phage display are 2 technologies commonly used for mAb discovery. For both technologies, antigen is required for immunization and for selection and screenings of antigen-specific clones.…”