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Case Studies

Antibody Discovery and Engineering for CCR8 Antibody with Cross-reactivity to Cyno Monkey CCR8

CCR8 is a GPCR highly expressed on Treg cells within tumors and becomes emerging target for immune oncology.

Due to the nature of GPCR and relatively lower sequence homology at extracellular regions between human and cyno monkey CCR8, it would be extremely difficult to identify CCR8-specific antibodies with cross-reactivity to cyno monkey CCR8.

In this case study, mice were immunized with HEK293 cells overexpressing human CCR8 for multiple times; then mice with decent serum titers to CCR8 were selected for tissue collection. B cells were isolated from the spleen cells collected from those immunized mice, and then used to generate phage libraries that can display antibodies in scFv fragment format. Since no qualified recombinant CCR8 protein was available, whole cell panning strategy was employed in which the phage libraries were enriched through multiple rounds of biopanning with CCR8-expressing cells and then screened for binding to human or cyno CCR8 cells by FACS. Multiple scFv clones with good binding activities to both human and cyno CCR8 were identified, and then converted to IgG format for functional characterization. The lead candidates also demonstrated good activities of inhibiting CCL1/CCR8 signaling pathway.

One of the lead candidates showed very strong binding activity to CCR8, its binding affinity values to human CCR8 cells and cyno CCR8 cells were 130 pM and 177 pM respectively, as determined by KinExA 4000 platform (Sapidyne Instruments Inc.). To further increase the binding affinity to CCR8, affinity maturation for the lead candidate was conducted through phage display technology. Four CDRs of the lead candidate were randomized with degenerated primers, resulting four mutagenic phage libraries displaying variants in scFv. Whole cell panning strategy was also employed to enrich these mutagenic phage libraries to capture the variants with increased binding activities. After analyzing the VH sequences of the variants with strong binding activities on FACS, the “hotspot” mutations from different CDRs which make critical impacts on antigen binding were identified. Then, the mutations from each CDR were also combined into new variants for further evaluation in the format of Fab or IgG. Dozens of variant IgGs were characterized by CCR8 binding and functional activity assays. The binding affinity to CCR8 of one variant was also measured by KinExA 4000 platform, which increased for 6 fold and reached to 20 pM.

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