A Novel Dual-Fc Bispecific Antibody with Enhanced Fc Effector Function.

Bispecific antibodies (BsAbs) are undergoing continued development for applications in oncology and autoimmune diseases. While increasing activity by having more than one targeting arm, most BsAb engineering employs single Fc engagement as monoclonal antibodies. Here, we designed a novel immunoglobulin gamma-1 (IgG1)-derived dual-Fc BsAb containing two Fc regions and two distinct asymmetric antigen binding arms comprising a Fab arm and another VHH domain. In conjunction with the knob-into-hole technology, dual-Fc BsAbs could be produced with a high yield and good stability. We explore how Fc engineering effects on dual-Fc constructs could boost the desired therapeutic efficacy. This new format enabled simultaneous bispecific binding to corresponding antigens. Furthermore, compared to the one-Fc control molecules, dual-Fc BsAbs were shown to increase the avidity-based binding to FcγRs to result in higher ADCC and ADCP activities by potent avidity via binding to two antigens and Fc receptors. Overall, this novel BsAb format with enhanced effector functionalities provides a new option for antibody-based immunotherapy.

Large scale controlled Fab exchange GMP process to prepare bispecific antibodies.

Objective: Bispecific antibodies (BsAbs) have demonstrated significant therapeutic impacts for the treatment of a broad spectrum of diseases that include oncology, auto-immune, and infectious diseases. However, the large-scale production of clinical batches of bispecific antibodies still has many challenges that include having low yield, poor stability, and laborious downstream purification processes. To address such challenges, we describe the optimization of the controlled Fab arm exchange (cFAE) process for the efficient generation of BsAbs. Methods: The process optimization of a large-scale good manufacturing practice (GMP) cFAE strategy to prepare BsAbs was based on screening the parameters of temperature, reduction, oxidation, and buffer exchange. We include critical quality standards for the reducing agent cysteamine hydrochloride. Results: This large-scale production protocol enabled the generation of bispecific antibodies with >90% exchange yield and at >95% purity. The subsequent downstream processing could use typical mAb procedures. Furthermore, we demonstrated that the bispecific generation protocol can be scaled up to ∼60 L reaction scale using parental monoclonal antibodies that were expressed in a 200 L bioreactor. Conclusion: We presented a robust development strategy for the cFAE process that can be used for a larger scale GMP BsAb production.

Anti-HBc IgG Responses Occurring at the Early Phase of Infection Correlate Negatively with HBV Replication in a Mouse Model.

Anti-HBc IgG is usually recognized as a diagnostic marker of hepatitis B, while the functional role anti-HBc IgG in HBV infection has not been fully elucidated. In this study, we firstly investigated the relationship between the anti-HBc IgG responses and the replication of HBV using AAV8-1.3HBV infected C57BL/6N mice. Our data showed that the anti-HBc IgG responses at the early phase of infection correlated negatively with the concentrations of circulating HBsAg and HBV DNA at both the early and chronic phases of infection. This observation was confirmed by an independent experiment using AAV8-1.3HBV infected C57BL/6J mice. Furthermore, to comprehend the potential causal relationship between the anti-HBc IgG responses and HBV infection, mice were treated with an anti-HBc monoclonal antibody at three days post AAV8-1.3HBV infection. Our data showed that the anti-HBc mAb significantly suppressed the fold increase of circulating HBsAg level, and the protective effect was not affected by NK cell depletion. Collectively, our study demonstrated that anti-HBc antibodies occurring at the early phase of HBV infection may contribute to the constraint of the virus replication, which might be developed as an immunotherapy for hepatitis B.

seq-ImmuCC: Cell-Centric View of Tissue Transcriptome Measuring Cellular Compositions of Immune Microenvironment From Mouse RNA-Seq Data.

The RNA sequencing approach has been broadly used to provide gene-, pathway-, and network-centric analyses for various cell and tissue samples. However, thus far, rich cellular information carried in tissue samples has not been thoroughly characterized from RNA-Seq data. Therefore, it would expand our horizons to better understand the biological processes of the body by incorporating a cell-centric view of tissue transcriptome. Here, a computational model named seq-ImmuCC was developed to infer the relative proportions of 10 major immune cells in mouse tissues from RNA-Seq data. The performance of seq-ImmuCC was evaluated among multiple computational algorithms, transcriptional platforms, and simulated and experimental datasets. The test results showed its stable performance and superb consistency with experimental observations under different conditions. With seq-ImmuCC, we generated the comprehensive landscape of immune cell compositions in 27 normal mouse tissues and extracted the distinct signatures of immune cell proportion among various tissue types. Furthermore, we quantitatively characterized and compared 18 different types of mouse tumor tissues of distinct cell origins with their immune cell compositions, which provided a comprehensive and informative measurement for the immune microenvironment inside tumor tissues. The online server of seq-ImmuCC are freely available at http://wap-lab.org:3200/immune/.

A human programmed death-ligand 1-expressing mouse tumor model for evaluating the therapeutic efficacy of anti-human PD-L1 antibodies.

Huge efforts have been devoted to develop therapeutic monoclonal antibodies targeting human Programmed death-ligand 1 (hPD-L1) for treating various types of human cancers. However, thus far there is no suitable animal model for evaluating the anti-tumor efficacy of such antibodies against hPD-L1. Here we report the generation of a robust and effective system utilizing hPD-L1-expressing mouse tumor cells to study the therapeutic activity and mode of action of anti-human PD-L1 in mice. The model has been validated by using a clinically proven hPD-L1 blocking antibody. The anti-hPD-L1 antibody treatment resulted in potent dose-dependent rejection of the human PD-L1-expressing tumors in mice. Consistent with what have observed in autochthonous mouse tumor models and cancer patients, the hPD-L1 tumor bearing mice treated by anti-hPD-L1 antibody showed rapid activation, proliferation and reinvigoration of the cytolytic effector function of CD8+T cells inside tumor tissues. Moreover, anti-hPD-L1 treatment also led to profound inhibition of Treg expansion and shifting of myeloid cell profiles, showing bona fide induction of multilateral anti-tumor responses by anti-hPD-L1 blockade. Thus, this hPD-L1 mouse model system would facilitate the pre-clinical investigation of therapeutic efficacy and immune modulatory function of various forms of anti-hPD-L1 antibodies.

Fusion-expressed CTB improves both systemic and mucosal T-cell responses elicited by an intranasal DNA priming/intramuscular recombinant vaccinia boosting regimen.

Previous study showed that CTB (Cholera toxin subunit B) can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting), pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562 ± 567 SFCs/10(6) splenocytes versus 330 ± 182 SFCs/10(6) splenocytes, P < 0.01), mesenteric LN (96 ± 83 SFCs/10(6) lymphocytes versus 1 ± 2 SFCs/10(6) lymphocytes, P < 0.05), draining LNs of respiratory tract (109 ± 60 SFCs/10(6) lymphocytes versus 2 ± 2 SFCs/10(6) lymphocytes, P < 0.01) and female genital tract (89 ± 48 SFCs/10(6) lymphocytes versus 23 ± 21 SFCs/10(6) lymphocytes, P < 0.01). These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.

Recruitment of HIV-1 target cells at topical mucosal sites: a sensitive and early marker for determining the safety of microbicide candidates.

To explore early biomarkers for establishing more sensitive safety evaluation assays in preclinical settings that determine the potential risks during the application of microbicide candidates, three representative microbicide candidates (cellulose sulphate, nonoxynol-9 and tenofovir), whose safety profiles have been well established in clinical trials, were included to gauge the sensitivities of different assays. Both mouse models and cell lines were employed to determine the sensitivities. The recruitment of immune cells at topical mucosal sites and the upregulation of HIV receptor/coreceptors in vitro were identified as highly sensitive biomarkers of the impact of microbicide candidates. Our data suggest that different evaluations/assays have their inherent sensitivities, and at least one assay from each sensitivity level should be included in the safety evaluation algorithm.

Efficacy, stability, and biosafety of sifuvirtide gel as a microbicide candidate against HIV-1.

Sifuvirtide is a proven effective HIV-1 entry inhibitor and its safety profile has been established for systemic administration. The present study evaluated the potential of sifuvirtide formulated in a universal gel for topical use as a microbicide candidate for preventing sexual transmission of HIV. Our data showed that sifuvirtide formulated in HEC gel is effective against HIV-1 B, C subtypes, CRF07_BC and CRF01_AE, the latter two recombinants represents the most prevalent strains in China. In addition, we demonstrated that sifuvirtide in gel is stable for at least 8 weeks even at 40°C, and did not cause the disruption of integrity of mucosal epithelial surface, or the up-regulation of inflammatory cytokines both in vitro or in vivo. These results suggest that sifuvirtide gel is an effective, safe and stable product, and should be further tested as a vaginal or rectal microbicide in pre-clinical model or clinical trial for preventing HIV sexual transmission.

Minocycline down-regulates topical mucosal inflammation during the application of microbicide candidates.

An effective anti-human immunodeficiency virus-1 (HIV-1) microbicide should exert its action in the absence of causing aberrant activation of topical immunity that will increase the risk of HIV acquisition. In the present study, we demonstrated that the vaginal application of cellulose sulfate (CS) gel induced topical mucosal inflammatory responses; the addition of minocycline to CS gel could significantly attenuate the inflammation in a mice model. The combined gel of CS plus minocycline not only reduced the production of inflammatory cytokines in cervicovaginal lavages (CVLs), also down-regulated the activation of CD4+ T cells and the recruitment of other immune cells including HIV target cells into vaginal tissues. Furthermore, an In vitro HIV-1 pseudovirus infection inhibition assay showed that the combined gel decreased the infection efficacy of different subtypes of HIV-1 pseudoviruses compared with that of CS gel alone. These results implicate that minocycline could be integrated into microbicide formulation to suppress the aberrant activation of topical mucosal immunity and enhance the safety profile during the application of microbicides.