L CD40-CD40L pathway is important in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. Engagement of the inhibitory Fcg-receptor (FcgR) IIb shows promise for in vivo antitumor activity of agonistic anti-CD40 monoclonal antibodies, and Fc region mutant designs of anti-CD40 antibodies had been discovered to enhance FcgRIIb engagement. With Twist’s precision DNA writing technologies, we have created phage display VHH and scFv libraries with diversity greater than 1 Å~ 1010 for optimal discovery. In this study, we performed high affinity binding of the antibodies by SPR and cell surface binding. The leads
are reformatted on human IgG2, IgG4, and IgG1 mutant. The in vitro properties of the CD40 agonistic antibodies demonstrate enhanced FcgRIIb engagement by NFkB activation. B cell activation is also detected by upregulation of CD86 and IL6 secretion. In humanized hCD40 mice model, we observed the engineered anti-CD40 agonists enhance anti-tumor immune function in vivo. These studies suggest that our antibodies can be potential drug for cancer immunotherapy.