Increasing stability of antibody via antibody engineering: stability engineering on an anti-hVEGF.

Antibody stability is very important for expression, activity, specificity, and storage. This knowledge of antibody structure has made it possible for a computer-aided molecule design to be used to optimize and increase antibody stability. Many computational methods have been built based on knowledge or structure, however, a good integrated engineering system has yet to be developed that combines these methods. In the current study, we designed an integrated computer-aided engineering protocol, which included several successful methods. Mutants were designed considering factors that affected stability and multiwall filter screening was used to improve the design accuracy. Using this protocol, the thermo-stability of an anti-hVEGF antibody was significantly improved. Nearly 40% of the single-point mutants proved to be more stable than the parent antibody and most of the mutations could be stacked effectively. The T50 also improved about 7°C by combinational mutation of seven sites in the light chain and three sites in the heavy chain. Data indicate that the protocol is an effective method for optimization of antibody structure, especially for improving thermo-stability. This protocol could also be used to enhance the stability of other antibodies.

Effective DNA epitope chimeric vaccines for Alzheimer's disease using a toxin-derived carrier protein as a molecular adjuvant.

Active amyloid-beta (Aß) immunotherapy is under investigation to prevent or treat Alzheimer disease (AD). We describe here the immunological characterization and protective effect of DNA epitope chimeric vaccines using 6 copies of Aß1-15 fused with PADRE or toxin-derived carriers. These naked 6Aß15-T-Hc chimeric DNA vaccines were demonstrated to induce robust anti-Aß antibodies that could recognize Aß oligomers and inhibit Aß oligomer-mediated neurotoxicity, result in the reduction of cerebral Aß load and Aß oligomers, and improve cognitive function in AD mice, but did not stimulate Aß-specific T cell responses. Notably, toxin-derived carriers as molecular adjuvants were able to substantially promote immune responses, overcome Aß-associated hypo-responsiveness, and elicit long-term Aß-specific antibody response in 6Aß15-T-Hc-immunized AD mice. These findings suggest that our 6Aß15-T-Hc DNA chimeric vaccines can be used as a safe and effective strategy for AD immunotherapy, and toxin-derived carrier proteins are effective molecular adjuvants of DNA epitope vaccines for Alzheimer's disease.

Rapid surveillance of New York City healthcare center egress behaviors during the 2020 COVID-19 lockdown.

This rapid response surveillance project was funded by the National Science Foundation (NSF) to collect "perishable" data on egress behaviors and neighborhood conditions surrounding healthcare centers (HCCs) in New York City (NYC) during the initial NYC COVID-19 PAUSE ordinance from March 22nd to May 19th, 2020. Anonymized data on NYC HCC egress behaviors were collected by observational field workers using phone-based mapping applications. Each egress trip record includes the day of week, time of day, destination category type, along with an array of behavioral outcome categories, ambient weather conditions and socio-economic factors. Egress trajectories with precise estimates of distance traveled and the spatial dispersion or "spread" around each HCC were added via post-processing. The data collection and cleaning process resulted in 5,030 individual egress records from 18 facilities over a 9-week period.

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-beta-like signaling pathway.

The four-and-a-half LIM (FHL) proteins belong to a family of LIM-only proteins that regulate cell proliferation, differentiation, and apoptosis. The exact functions of each FHL protein in cancer development and progression remain unknown. Here we report that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, in a TGF-beta-independent manner. Casein kinase 1delta, but not the TGF-beta receptor, was required for the FHL-mediated TGF-beta-like responses, including increased phosphorylation of Smad2/3, interaction of Smad2/3 and Smad4, nuclear accumulation of Smad proteins, activation of the tumor suppressor gene p21, and repression of the oncogene c-myc. FHL1-3 inhibited anchorage-dependent and -independent growth of a human hepatoma cell line in vitro and tumor formation in nude mice. Further analysis of clinical samples revealed that FHL proteins are often downregulated in hepatocellular carcinomas and that this correlates with decreased TGF-beta-like responses. By establishing a link between FHL proteins and Smad proteins, this study identifies what we believe to be a novel TGF-beta-like signaling pathway and indicates that FHL proteins may be useful molecular targets for cancer therapy.

Regulatory utility of pharmacometrics in the development and evaluation of antimicrobial agents and its recent progress in China.

Pharmacometrics is an emerging science that interprets drug, disease, and trial information in a mathematical fashion to inform and facilitate efficient drug development and/or regulatory decisions. Pharmacometrics study is increasingly adopted in the regulatory review of new antimicrobial agents. We summarized the 31 antimicrobial agents approved by the US Food and Drug Administration (FDA) and the 26 antimicrobial agents approved by European Medicines Agency (EMA) from January 2001 to May 2019. We also reviewed recent examples of utilizing pharmacometrics to support antimicrobial agent's registration in China, including modeling and simulation methods, effects of internal/external factors on pharmacokinetic (PK) parameters, safety and efficacy evaluation in terms of exposure-response analysis, refinement of the wording of product labeling and package leaflet, and possible postmarketing clinical trial. Ongoing communication among regulator, academia, and industry regarding pharmacometrics is encouraged to streamline and facilitate the development of new antimicrobial agents. The industry can maximize its benefit in drug development through continued pharmacometrics education/training.

PRSS contributes to cetuximab resistance in colorectal cancer.

Cetuximab improves the survival of patients with metastatic colorectal cancer. The main limitation is primary and secondary resistance, the underlying mechanism of which requires extensive investigation. We proved that PRSS expression levels are significantly negatively associated with the sensitivity of cancer cells to cetuximab. Detailed mechanistic analysis indicated that PRSS can cleave cetuximab, leading to resistance. Cetuximab or bevacizumab combined with SPINK1, a PRSS inhibitor, inhibited cell growth more efficiently than cetuximab or bevacizumab alone in xenograft models. PRSS levels in the serum of 156 patients with mCRC were analyzed, and poor efficacy of cetuximab therapy was observed in patients with aberrant PRSS expression. PRSS expression in monoclonal antibody (mAb)-treated patients with cancer from The Cancer Genome Atlas database was also evaluated to determine whether patients with higher PRSS expression have significantly reduced progression-free survival. Our work provides a strong scientific rationale for targeting PRSS in combination with cetuximab therapy.

A Novel Anti-EGFR mAb Ame55 with Lower Toxicity and Better Efficacy than Cetuximab When Combined with Irinotecan.

To improve efficacy and minimize toxicity of EGFR inhibition treatment, we developed Ame55, a novel anti-EGFR IgG1 with lower affinity to EGFR than cetuximab (C225) from a human phage library. Ame55 had lower bioactivity than cetuximab in vitro but similar antitumor efficacy as cetuximab in vivo. Moreover, Ame55 was more efficacious than cetuximab in a Lovo cell xenograft tumor model when combined with irinotecan (CPT-11). Ame55 concentrates in the mouse xenograft tumor and has less toxicity than cetuximab in cynomolgus monkeys in an overdose study.

A novel fully human anti-CD47 antibody as a potential therapy for human neoplasms with good safety.

Strategies for targeting CD47 are becoming a hot spot of cancer immunotherapy. However the ubiquitous expression of CD47, especially on the RBC, makes the targeted therapy facing safety risk issues. So, how to balance the safety and efficacy during CD47 inhibition is currently a major question. We had reported an anti-CD47 antibody ZF1 with potent anti-tumor effect. In this study, we further developed and assessed a novel fully human anti-CD47 antibody, AMMS4-G4, derived from ZF1 using affinity maturation. AMMS4-G4 exhibited equivalent anticancer effects with Hu5F9-G4, a humanized anti-CD47 antibody in clinical trial, on the potential of inducing significant phagocytosis of tumor cells in vitro and prolonging the survival of leukemia xenografted mice. Additionally, AMMS4-G4 significantly inhibited the growth of grafted solid tumors by enhancing macrophage infiltration and modestly enhanced the anti-tumor activity of opsonizing antibody and antiangiogenic therapy. In cynomolgus monkeys, AMMS4-G4 was safely administered, was well tolerated at doses of 30 and 60 mg/kg, and did not produce serious adverse events, except for the reversible anemia, which was observed after 3 days and started to recover from 9 days later. Remarkably, it was proved by in vitro assay that Hu5F9-G4 induced RBC hemagglutination which wasn't observed in AMMS4-G4. On the whole, AMMS4-G4 was demonstrated to be a promising candidate with great potential and safe profile for cancer immunotherapy.

A fully human anti-CD47 blocking antibody with therapeutic potential for cancer.

CD47/SIRPα interaction serves as an immune checkpoint for macrophage-mediated phagocytosis. Mouse anti-CD47 blocking antibodies had demonstrated potent efficacy in the treatment of both leukemic and solid tumors in preclinical experimentations, and therefore had moved forward rapidly into clinical trials. However, a fully human blocking antibody, which meets clinical purpose better, has not been reported for CD47 up to date. In this study, we reported the isolation of a fully human anti-CD47 blocking antibody, ZF1, from a phage display library. ZF1 displayed high specificity and affinity for CD47 protein, which were comparable to those for humanized anti-CD47 blocking antibody B6H12. Importantly, ZF1 treatment could induce robust, or even stronger than B6H12, phagocytosis of leukemic cancer cells by macrophage in vitro, and protect BALB/c nude mice from cancer killing by engrafted leukemic cells (CCRF and U937) to a similar extent as B6H12 did. Thus, these data provide primary early pre-clinical support for the development of ZF1 as a fully human blocking antibody to treat human leukemia by targeting CD47 molecule.

A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic, heterogeneous autoimmune disease short of effective therapeutic agents. A multitude of studies of SLE in the last decade have accentuated a central role of the interferon alpha (IFN-α) pathway in SLE pathogenesis. We report here a candidate therapeutic neutralizing antibody, AIA22, with a different binding epitope and discrepant neutralizing profile from the anti-multiple IFN-α subtype antibodies currently in clinical trials. AIA22 specifically interacts with multiple IFN-α subtypes, binds to the type I IFN receptor 2 (IFNAR2) recognition region of IFN-α (considered a novel antigen epitope), and effectively neutralizes the activity of almost all of the IFN-α subtypes (with the exception of IFN-α7) both in vitro and in vivo. Concurrently, structural modeling and computational design yielded a mutational antibody of AIA22, AIAmut, which exhibited substantially improved neutralizing activity to multiple IFN-α subtypes.

RSRC1 SUMOylation enhances SUMOylation and inhibits transcriptional activity of estrogen receptor ß.

The transcription factor estrogen receptor ß (ERß) plays roles in the central nervous, endocrine, cardiovascular, and immune systems. ERß can be SUMOylated. However, the underlying mechanism remains unclear. Here, we show that RSRC1/SRrp53 interacts with ERß and SUMOylation of RSRC1 is required for regulation of PIAS1-mediated ERß SUMOylation. RSRC1 promotes ERß SUMOylation through enhanced interaction between ERß and PIAS1. RSRC1 represses ERß transcriptional activity through regulation of ERß SUMOylation. By establishing RSRC1 as a novel cofactor for SUMOylation, our data provide insight into regulation of ERß SUMOylation and indicate that SUMOylation of one protein can regulate another protein SUMOylation.

Strikingly reduced amyloid burden and improved behavioral performance in Alzheimer's disease mice immunized with recombinant chimeric vaccines by hexavalent foldable Aß1₋15 fused to toxin-derived carrier proteins.

Targeting on the amyloid-ß (Aß) is a promising immunotherapeutic strategy for Alzheimer's disease (AD). However, Aß(1-15) sequence alone induces low antibody response and poor protection against AD. We describe here the immunological characterization and protective efficacy of several recombinant chimeric vaccines with hexavalent foldable Aß(1-15) (6Aß15) fused to PADRE or toxin-derived carrier proteins. Immunization with these chimeric antigens generated robust Th2 immune responses with high anti-Aß42 antibody titers in different mice, which recognized neurotoxic Aß42 oligomers, but did not stimulate Aß42-specific T cell responses. These 6Aß15 chimeric vaccines markedly reduced Aß pathology and prevented development of behavioral deficits in immunized older AD mice. Importantly, toxin-derived carrier proteins as molecular adjuvants of chimeric vaccines could substantially boost immune responses and overcome Aß- and old age-associated hypo-responsiveness, and elicit long-term Aß-specific antibody response, which in turn inhibited Aß-mediated pathology and improved acquisition and retention of spatial memory in immunized AD mice. These data indicate that toxin fragments as molecular adjuvants are promising new tools for the rational design and development of prototype chimeric vaccines for AD and this type of chimeric vaccine design has the added advantage of overcoming hypo-responsiveness in elderly AD patients with pre-existing memory Th cells from tetanus toxin.