Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions.

Siglec-15, an inhibitory immune checkpoint, is an emerging target in cancer immunotherapy. Blocking the function of Siglec-15 is an excellent strategy for cancer treatment and antibody blockade has been used to target Siglec-15. However, whether Fc-mediated effector functions contribute to the therapeutic effect of antibodies remains unclear. Herein, we generated a monoclonal antibody, 1-15D1, which had a high binding affinity with Siglec-15 and strongly activated T-cell immune response in vitro. Subsequently, the Fc-mediated effector functions of 1-15D1 were explored in a Siglec-15 humanized mouse model, and further improvement in antitumor efficacy was observed in the mouse IgG2a isotype group. Thus, we demonstrate that the antitumor effects of 1-15D1 were mediated via multiple factors. In addition to the T-cell immune response, 2 novel mechanisms were explored, including the internalization of the cell surface Siglec-15 and Fc-mediated effector functions. In conclusion, our studies not only provide a potential agent for the improvement of cancer immunotherapy but also suggest that a specific role of Fc-mediated immune regulation may improve the therapeutic potency of Siglec-15 monoclonal antibody.

Structural basis of SALM5-induced PTPδ dimerization for synaptic differentiation.

SALM5, a synaptic adhesion molecule implicated in autism, induces presynaptic differentiation through binding to the LAR family receptor protein tyrosine phosphatases (LAR-RPTPs) that have been highlighted as presynaptic hubs for synapse formation. The mechanisms underlying SALM5/LAR-RPTP interaction remain unsolved. Here we report crystal structures of human SALM5 LRR-Ig alone and in complex with human PTPδ Ig1-3 (MeA-). Distinct from other LAR-RPTP ligands, SALM5 mainly exists as a dimer with LRR domains from two protomers packed in an antiparallel fashion. In the 2:2 heterotetrameric SALM5/PTPδ complex, a SALM5 dimer bridges two separate PTPδ molecules. Structure-guided mutations and heterologous synapse formation assays demonstrate that dimerization of SALM5 is prerequisite for its functionality in inducing synaptic differentiation. This study presents a structural template for the SALM family and reveals a mechanism for how a synaptic adhesion molecule directly induces cis-dimerization of LAR-RPTPs into higher-order signaling assembly.

[Preparation, identification and application of monoclonal antibody against urease subuint B of Helicobacter pylori].

OBJECTIVE: To prepare and characterize the monoclonal antibody (mAb) against Helicobacter pylori (Hp) and establish a competitive ELISA used for detection of Hp antibodies in the sera of Hp-infected patients. METHODS: BALB/c mice were immunized with inactivated Hp to generate Hp mAb using the hybridoma technology. Hp mixed proteins including cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA) and urease, as well as inactivated Hp were applied to screen positive hybridoma. Selected Hp mAb was analyzed and characterized with ELISA and Western blotting, and then labeled with horseradish peroxidase (HRP) for establishing a competitive ELISA to detect Hp antibodies in the sera of Hp-infected patients. RESULTS: One Hp mAb named as C3 was selected after screening large amount of hybridoma, and the C3 Hp mAb was special for IgG2a subtype with the affinity titer of 1×10(7). Western blotting, ELISA and mass spectrum analysis indicated that the C3 Hp mAb could recognize Hp urease subunit B specifically. Using the C3 Hp mAb, we developed a competitive ELISA which could be used to detect Hp antibodies in the sera of Hp-infected patients. CONCLUSION: We successfully obtained one mAb that could specifically recognize Hp urease subunit B and developed a competitive ELISA using the Hp mAb.