Next generation of anti-PD-L1 Atezolizumab with enhanced anti-tumor efficacy in vivo.

FDA-approved anti-PD-L1 antibody drug Atezolizumab is a human IgG1 without glycosylation by an N297A mutation. Aglycosylation of IgG1 has been used to completely remove the unwanted Fc-mediated functions such as antibody-dependent cytotoxicity (ADCC). However, aglycosylated Atezolizumab is very unstable and easy to form aggregation, which causes quick development of anti-drug antibody (ADA) in 41% of Atezolizumab-treated cancer patients, eventually leading to loss of efficacy. Here, we report the development of the anti-PD-L1 antibody drug Maxatezo, a glycosylated version of Atezolizumab, with no ADCC activity, better thermo-stability, and significantly improved anti-tumor activity in vivo. Using Atezolizumab as the starting template, we back-mutated A297N to re-install the glycosylation, and inserted a short, flexible amino acid sequence (GGGS) between G237 and G238 in the hinge region of the IgG1 heavy chain. Our data shows that insertion of GGGS, does not alter the anti-PD-L1's affinity and inhibitory activity, while completely abolishing ADCC activity. Maxatezo has a similar glycosylation profile and expression level (up to 5.4 g/L) as any normal human IgG1. Most importantly, Maxatezo's thermal stability is much better than Atezolizumab, as evidenced by dramatic increases of Tm1 from 63.55 °C to 71.01 °C and Tagg from 60.7 °C to 71.2 °C. Furthermore, the levels of ADA in mice treated with Maxatezo were significantly lower compared with animals treated with Atezolizumab. Most importantly, at the same dose (10 mg/kg), the tumor growth inhibition rate of Maxatezo was 98%, compared to 68% for Atezolizumab.

Novel strategy for expression and characterization of rabies virus glycoprotein.

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few µg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection.