Generation and chracterisation of anti-LILR antibodies for immunotherapy

Leukocyte Immunoglobulin (Ig)-Like Receptors (LILRs) (LIRs/ILT/CD85) are a family of cell surface receptors involved in innate and adaptive immunity. There are six activatory (LILRA) and five inhibitory (LILRB) LILRs, with imbalances associated with the progression of autoimmune diseases such as rheumatoid arthritis. The inhibitory LILRs are up-regulated in anti-inflammatory environments and have been implicated in tumour progression. LILRs could therefore be potential immunotherapeutic targets to treat both cancer and autoimmunity.LILRB3 (ILT5/CD85a) is an inhibitory receptor belonging to this family. Although LILRB1 and LILRB2 have been extensively studied, LILRB3 has been less so. Its function is unclear but its restricted expression profile on myeloid cells makes it an attractive target. To help establish the potential of this family of receptors as targets for immunotherapy, a panel of LILRB1-, LILRB2- and LILRB3-specific monoclonal antibodies (mAbs) were generated by propriety phage display technology. To confirm specificity the mAbs were assessed for binding to LILRB-transfectants compared to mock-transfectants, as well as cells expressing other related LILRs. Two, six and sixteen antibodies displayed specific binding to LILRB1, LILRB2 and LILRB3, respectively in these assays. Antibody binding to LILRBs on myeloid cells including monocytes, macrophages and neutrophils were confirmed. Fine specificity and epitope mapping was performed using cross-blocking assays and HEK 293F-transfectants expressing mutated molecules of LILRB3, displaying varying numbers of extracellular domains. Using reporter cells capable of detecting receptor cross-linking, the antibodies were then screened for their ability to activate or inhibit cellular activation. The antibodies were assessed for their ability to regulate certain aspects of myeloid cell biology, including regulation of T-cell proliferation and macrophage phagocytosis. Finally, the antibodies were tested in vivo to assess their ability to act as direct targeting antibodies. These reagents allowed us to assess the function and immunotherapeutic potential of LILR mAbs