Background T regulatory cells (Tregs) are potent suppressors of immune activation in the periphery and tumor microenvironment (TME). Tumor-infiltrating Tregs have also been associated with resistance to cancer therapies. Loss of peripheral Tregs can lead to widespread autoimmunity and tissue destruction; therefore, specifically depleting tumor Tregs is an attractive therapeutic approach to locally activate the immune system. CCR8 expression is highly restricted to tumor Tregs across multiple cancer types, supporting the notion that CCR8 targeting may induce tumor-specific Treg depletion while sparing peripheral Tregs. Moreover, depletion of CCR8+ Tregs leads to significant tumor growth inhibition with correlative tumor Treg depletion in established CT-26 tumors. These data provide rationale for targeting CCR8 to deplete tumor Tregs. Here, we describe the development of SRF114, a fully human IgG1 anti-CCR8 antibody that induces tumor Treg destruction through antibody-dependent cellular cytotoxicity (ADCC).
Methods Virus panning against the N-terminal region of CCR8 and subsequent affinity maturation process led to discovery of SRF114, a fully human monoclonal antibody that is specific to CCR8. To evaluate SRF114 specificity, binding was profiled on recombinant CCR8 N-terminus, CCR8+ and CCR8- cell lines, and primary cell cultures. An extracellular protein target cell microarray was used to further validate specificity. SRF114 functional assays included the Promega CD16 (V/F variants) ADCC signaling assay, PBMC/293T-hCCR8+ cell co-culture experiments, and natural killer (NK)-activation assays targeting Raji-CCR8+ cell lines. To confirm tumor Treg binding and depletion, NK allogenic co-culture experiments were performed with SRF114 using isolated tumor infiltrating lymphocyte cultures from freshly resected tumors.
Results A tumor Treg-restricted pattern of CCR8 expression was confirmed using publicly available datasets and profiling of CCR8 expression on Tregs from fresh tumor tissues. SRF114 binds to CCR8-expressing 293T cells with pM affinity and not to parental cells. SRF114 does not bind any cell populations in PBMCs from healthy donors and has no other protein targets assessed by cell microarray. In dose-dependent ADCC assays, SRF114 induces cell killing with pM EC50 values, which is further enhanced by removing the fucose groups from the Fc-domain. Finally, SRF114 specifically binds to human tumor Tregs and induces killing of Tregs in NK co-culture experiments.
Conclusions The fully human anti-CCR8 antibody SRF114 specifically binds to and targets CCR8+tumor Tregs for depletion, likely through ADCC. Through this mechanism, SRF114 treatment may alter the TME to support immune destruction of human tumors.
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