Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.

Multi-parametric analysis reveals enhanced G2-phase arrest of an optimized anti-HER2 antibody to inhibit breast cancer.

OBJECTIVES: To find a "me-better" antibody by epitope-specific antibody optimization and multi-parametric analysis. RESULTS: Using epitope-specific library based on the commercial drug, Pertuzumab/2C4, we screened a novel human anti-HER2 antibody, MIL5, which has slightly higher affinity than the drug. MIL5 and 2C4 share the same epitope to bind HER2; however, MIL5 bound to HER2 His235-His245 more tightly than 2C4, which could be the main reason of its enhanced affinity. In vivo experiments also showed MIL5 had stronger anti-cancer activity than 2C4; however, the classical flow cytometry assays to detect cell apoptosis or cycling did not show convincing evidence of the advantages of MIL5. Thus we introduced the multi-parameter in-cell analysis method to evaluate the superiority of MIL5 to 2C4 in arresting cancer cells in G2-phase to inhibit cell growth and/or proliferation. CONCLUSION: Multi-parametric method confirmed stronger arrest of G2 by MIL5 to show better anti-cancer function both in vitro and in vivo than 2C4.

A novel anti-TNF scFv constructed with human antibody frameworks and antagonistic peptides.

The introduction of TNF inhibitors has revolutionized the treatment of some chronic inflammatory diseases, e.g., rheumatoid arthritis and Crohn's disease. However, immunogenicity is one of the important mechanisms behind treatment failure, and generally, switching to another TNF inhibitor will be the first choice for patients and doctors, which results in unmet need for novel anti-TNF agents. Small antibody molecules with less number of epitope may be valuable in less immunogenicity. In this study, with the help of computer-guided molecular design, single-chain variable fragment (scFv) TSA2 was designed using consensus frameworks of human antibody variable region as scaffold to display anti-TNF antagonistic peptides. TSA2 showed evidently improved bioactivity over TSA1 (anti-TNF scFv explored before) and almost similar activity as S-Remicade (the scFv form of Remicade, anti-TNF antibody approved by FDA), especially in inhibiting TNF-induced cytotoxicity and NF-κB activation. Human antibody consensus frameworks with less immunogenicity have been used in the designing of VH domain antibody, scFv TSA1 and TSA2. A serial of TNF-related works convinced us that the novel design strategy was feasible and could be used to design inhibitors targeting more other molecules than TNF-α. More importantly, these designed inhibitors derived from computer modeling may form a virtual antibody library whose size depends on the number of candidate antagonistic peptides. It will be molecular-targeted virtual antibody library because of the specific antagonistic peptides and the potential antibodies could be determined by virtual screening and then confirmed by biologic experiments.

Improving Trastuzumab's Stability Profile by Removing the Two Degradation Hotspots.

Stability of recombinant monoclonal antibodies (mAbs) is essential for their clinical application. The presence of the two degradation hotspots, namely, LC-Asn30 and HC-Asp102, in its complementary determinant regions prevents trastuzumab (Herceptin®) from being supplied in a drug product format of liquid formulation. To improve the stability, a new antibody was created by replacing the two residues with chemically similar amino acids of LC-Gln30 and HC-Glu102. This new mAb, named as T-mAb2, exhibited a simple and more uniform charge heterogeneity profile than T-mAb1, which is trastuzumab made in our laboratory, as displayed by the difference between their main peak area percentages (82.9% for T-mAb2 vs. 60.5% for T-mAb1). Computer modeling results, physicochemical and biological characterization, and stability profiling studies on T-mAb2 and T-mAb1 demonstrated that stability of T-mAb2 was significantly improved. In comparison with T-mAb1, although its in vitro human epidermal growth factor receptor 2 (HER2)-target binding activities were reduced slightly, in vivo tumor growth inhibiting activity was not affected, as demonstrated by the study results using the SKOV3 xenograft mouse model. Hence, a new anti-HER2 antibody was generated with improved stability that could be used to produce the drug product in liquid formulation for cost saving and more convenient usage.

Humanization of an anti-CCR4 antibody that kills cutaneous T-cell lymphoma cells and abrogates suppression by T-regulatory cells.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17- and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4(+) CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitro mechanistic studies showed that both complement-dependent cytotoxicity (CDC) and neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) likely mediated this effect. mAb1567 also exerts human NK cell-mediated ADCC activity in vitro. Moreover, mAb1567 also effectively inhibits chemotaxis of CD4(+)CD25(high) Tregs via CCL22 and abrogates Treg suppression activity in vitro. An affinity-optimized variant of humanized mAb1567, mAb2-3, was selected for further preclinical development based on its higher binding affinity and more potent ADCC and CDC activities. Taken together, this high-affinity humanized mAb2-3 with potent antitumor effect and a broad range of mechanisms of action may provide a novel immunotherapy for CTCL and other solid tumors.

Identification of conformational core epitope Lys68 in C5a based on the 3-D modeling complex C5a and its functional antibody F20.

Inhibition of C5a by antibodies has been demonstrated to dramatically improve survival in various sepsis models in mice and rats. The structural basis of C5a mediated bioactivity and C5a antibody mediated neutralization are of interesting to be investigated. In the previous study, we obtained a novel functional mouse antibody named as F20. With computer-guided modeling method, the 3-D theoretical structure of F20 Fv fragment was constructed. Using the crystal structure of C5a, the 3-D complex structure of C5a and F20 Fv fragment was modeled with molecular docking method. Based on distance geometry method and intermolecular interaction theory, the key residue Lys(68) in C5a identified by F20 was predicted. The mutant experimental results showed that the residue Lys(68) was the critical residue of C5a for it's bioactivity and F20 binding activity. The present study shed new light on the structural basis of C5a mediated bioactivity. The identification of the critical residue will provide useful information for human complement C5a targeted therapeutic intervention.

The N- and C-terminal carbohydrate recognition domains of galectin-9 contribute differently to its multiple functions in innate immunity and adaptive immunity.

By binding to T cell Ig mucin-3 (Tim-3) expressed on different cells, galectin-9 (Gal-9) mediates two important functions, triggering T cell death and activating innate immune cells. The mechanisms by which ligation of the same molecule on different cell types mediates different effects are largely unclear. Gal-9 contains two carbohydrate recognition domains (CRD) in the N- and C-terminal regions (Gal-9-N and Gal-9-C). The N and C terminals of Gal-9 have been shown to have different activities in promoting T cell death. However, whether the differences between two domains account for its dual functions remains to be elucidated. Here we hypothesized that the different functions of Gal-9 in innate immunity and adaptive immunity are mediated by different domains. To test this, we created recombinant Gal-9 (Gal-9-NC) and homodimers containing either the NCRD (Gal-9-N) or the CCRD (Gal-9-C). All these Gal-9 constructs can activate dendritic cells (DCs) and induce T cell death. However, the Gal-9-C was much more potent than the Gal-9-N in inducing T cell death, while the Gal-9-N was much more effective in activating DCs by inducing much higher TNF-α and IL-6 production, greater phosphorylation of p38 and AKT. In both DC and T cells, Gal-9-N but not Gal-9-C stimulation resulted in markedly iκBα degradation. Finally, computer analyses suggested different patterns and affinities for the binding of the Gal-9-N and Gal-9-C to their receptor, Tim-3. Our data suggest that the N- and C-terminal CRDs of Gal-9 contribute differently to its ability to induce T cell death and to activate DCs. Further investigations on the underlying mechanisms will provide new insights into the biochemical basis for the multiple activities of Gal-9.

Molecular modeling and affinity determination of scFv antibody: proper linker peptide enhances its activity.

One of existing strategies to engineer active antibody is to link V(H) and V(L) domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or single-chain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned V(H) and V(L) domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and approximately 24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

Overexpression, effective renaturation, and bioactivity of novel single-chain antibodies against TNF-alpha.

Neutralization of tumor necrosis factor-alpha (TNF-alpha) has become an effective therapeutic strategy for TNF-related autoimmune diseases. Due to the limitations of the large molecular inhibitors in the therapy, development of novel TNF-alpha inhibitors is very attractive and useful. In this study, based on the previously designed domain antibody, two novel human anti-TNF single-chain antibodies were constructed using modular consensus frameworks of human antibody as scaffold to display the antagonistic peptides. A variety of expression plasmids were used to determine the optimal expression system. The single-chain antibodies were always overexpressed in E.coli BL21(DE3) host as inclusion bodies. Under the optimized refolding conditions, the inclusion bodies were renatured successfully and the refolded single-chain antibodies could bind with TNF-alpha and block TNF-induced cytotoxicity on L929 cells. The bioactivity of the single-chain antibodies was significantly increased over the domain antibody.

Human IgG1 Cgamma1 domain is crucial for the bioactivity of the engineered anti-CD20 antibodies.

In this study, we discussed the necessity of human IgG1 Cgamma1 domain for recombinant antibody using computer-aided homology modeling method and experimental studies. The heavy (VH) and light (VL) chain variable regions of 1-28, a murine IgM-type anti-CD20 mAb, were ligated by linker peptide (Gly4Ser)3 to form the single-chain Fv fragment (scFv). Then, the engineered antibody (LH1-3) was generated by fusing scFv with the entire IgG1 heavy constant regions. The 3-D structure of LH1-3 was modeled using computer-aided homology modeling method and the binding activity of LH1-3 was evaluated theoretically. Compared to the 3-D structure of the Fv fragment of the parent antibody, the conformation of the active pocket of LH1-3 was remained because of the rigid support of Cgamma1. Further experimental results of flow cytometry showed that the engineered anti-CD20 antibody possessed specifically binding activity to CD20-expressing target cells. The anti-CD20 antibody fragments could also mediate complement-dependent cytotoxicity (CDC) of human B-lymphoid cell lines. Our study highlights some interests and advantages of a methodology based on the homology modeling and analysis of molecular structural properties.