726 SRF114 is a fully human, CCR8 selective IgG1 antibody that induces destruction of tumor Tregs through ADCC

BackgroundT 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).MethodsVirus 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.ResultsA 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.ConclusionsThe 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.

Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0–20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

New Advances in Cell Microarray Technology to Expand Applications in Target Deconvolution and Off-Target Screening

Identifying the cell surface receptors of natural ligands, and deconvoluting the receptor targets of candidate drug leads, presents a challenge in medical research and drug discovery. Traditionally, success rates have been low, and screening efforts often generate numerous false-positive hits that require extensive follow-up to either validate or disregard. If successful, receptor identification enables the discovery of previously unknown, disease-relevant targets, provides critical insights into biological pathways and disease processes, and allows for secondary targets to be uncovered. By expressing the majority of the human plasma membrane proteome in human cells on glass slides in situ, human cell microarray technology provides a powerful approach for identifying receptor target interactions. This approach significantly increases the success rates in identifying specific primary receptor targets and off-targets while limiting the number of false-positive hits. Here we describe cell microarray technology, focusing on new advances including the use of whole cells as bait for receptor interactions, and the inclusion of secreted proteins that widens the utility of the technology in off-target screening.