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.

In vivo Screen Identifies Zdhhc2 as a Critical Regulator of Germinal Center B Cell Differentiation.

Germinal center (GC) B cell differentiation is critical for the production of affinity-matured pathogen-specific antibodies, the dysregulation of which may lead to humoral immunodeficiency or autoimmunity. The development of an in vivo screening system for factors regulating GC B cell differentiation has been a challenge. Here we describe a small-scale in vivo screening system with NP-specific B1-8hi cells and a retroviral shRNA library targeting 78 candidate genes to search for B cell-intrinsic factors that specifically regulate GC B cell differentiation. Zdhhc2, a gene encoding palmitoyltransferase ZDHHC2 and highly expressed in GC B cells, is identified as a strong positive regulator of GC B cell differentiation. B1-8hi cells transduced with Zdhhc2-shRNA are severely compromised in differentiating into GC B cells. A further analysis of in vitro differentiated B cells transduced with Zdhhc2-shRNA shows that Zdhhc2 is critical for the proliferation and the survival of B cells stimulated by CD40L, BAFF, and IL-21 and consequently impacts on their differentiation into GC B cells and post-GC B cells. These studies not only identify Zdhhc2 as a novel regulator of GC B cell differentiation but also represent a proof of concept of in vivo screen for regulators of GC B cell differentiation.

Rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal antibodies against DKK2 for cancer therapy.

Dickkopf-related protein 2 (DKK2)is a member of the Dickkopf family in Wnt signaling pathway. Recently, we found that antibodies against DKK2 could activate natural killer (NK) and CD8+ T cells in tumors and inhibit tumor growth. In this paper, we report the rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal anti-DKK2 antibodies. To break the immune tolerance, we designed and chemically synthesized six peptides corresponding to different regions of DKK2 as immunogens and found five of them could generate mouse polyclonal antibodies that can bind to the active recombinant human DKK2 protein. Neutralizing mouse monoclonal antibodies (5F8 and 1A10) against human DKK2 were successfully developed by immunizing the mice with two different peptides (34KLNSIKSSL42 and 240KVWKDATYS248) conjugated to Keyhole limpet hemocyanin (KLH). The monoclonal antibodies not only abolish DKK2's suppression of Wnt signaling in vitro but also inhibits tumor growth in vivo. Currently, those two mAbs are undergoing humanization as immunotherapy candidates and may offer a new drug for treatment of human cancers.

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.

Production of monoclonal antibodies for measuring Avastin and its biosimilar by Sandwich ELISA.

The development of Bevacizumab (Avastin) biosimilar products has grown rapidly over the last ten years as the original Avastin's patent will expire soon. The approval of Avastin biosimilars requires the demonstration of similarity between the biosimilar and the reference product. To support pre-clinical and clinical studies, pharmacokinetic (PK) assays are required to measure the biosimilar and Avastin with comparable precision and accuracy. The PK assay of Avastin employed by Genentech was a Sandwich ELISA which could detect the total drug concentration. However, it was developed in-house and not commercially available. Therefore, in most of the Avastin biosimilar pre-clinical studies, the antibody drug concentrations were measured using an indirect ELISA against coated VEGF, which could only measure the free instead of the total antibody drugs. It failed the essential requirement to develop the biosimilars. In this study, we reported the generation of mouse monoclonal antibodies (mAbs) that specifically recognize Avastin in a VEGF non-competitive manner. Using a pair of non-VEGF competing anti-Avastin mAbs, a Sandwich ELISA was developed with a lower limit of quantitation (LLOQ) at 400 ng/mL and upper limit of quantitation (ULOQ) at 12800 ng/mL. The assay validation was carried out with serum samples from monkey treated with Avastin biosimilar at seven different time points. Our data showed that the Sandwich ELISA kit we developed is sensitive, simple, reproducible and ready for use in human clinical trials.