G-Coupled Protein Receptors (GPCR) remain one of the most attractive but more challenging therapeutic targets for antibody discovery. Complement component 5a receptor 1 (C5aR1) is a GPCR of interest with potential therapeutic applications in immune-oncology and inflammation. This GPCR is involved with the complement cascade and in innate immunity. Stimulation of C5aR1 by its ligand, C5a, promotes tumorigenesis by suppressing T-cell mediated anti-tumor activity while blockade of C5aR signaling has been shown to attenuate tumor growth. In a combined approach with Macromoltek, we leverage Twist’s DNA synthesis and library assembly technology with structural-based machine learning to design a targeted anti-C5aR discovery library to identify several highly potent and selective lead antibodies against C5aR. Several of the candidates identified from this study are also shown to have functionally antagonistic effects on C5aR1-mediated signaling in cellbased assays. This work demonstrates how the combination of highly specific DNA synthesis, coupled with informed design, can be paired to successfully prosecute challenging targets for novel therapeutic discovery.

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.

Promising therapeutic antibodies from other species typically go through the process of humanization before they reach human patients. Humanization involves altering its amino acid sequence such that it more closely resembles a human antibody. While this is a common method used to minimize immunogenicity risk, humanization remains a substantial technical barrier. Twist Bioscience has developed a humanization platform that combines highly precise DNA synthesis with machine learning (ML) to improve the process to enable faster, more efficient, and ultimately more productive antibody engineering.