Protein and Peptide Letters - Volume 24, Issue 12, 2017

Background: Bispecific antibodies, BsAbs, are molecules with the ability to bind to two different epitopes on the same or different antigens. c-MET, cellular-mesenchymal to epithelial transition factor, is deregulated in many types of human malignancies. Abnormal c-MET activation in cancer correlates with poor prognosis. PD-1, programmed death-1, is an additional inhibitory receptor expressed by T cells. Blocking the interactions between PD-1 and PD-L1 has emerged as a promising immunotherapy for treating cancer. Objectives: The goal of this study was to identify a novel bispecific antibody targeting both c-MET and PD-1 as an anti-cancer therapeutic candidate. Methods: The BsAb was produced using 293E expression system and purified by Protein A affinity chromatography. Then the binding specificity and affinity of the BsAb was examined by FACS and biolayer light interferometry. The ability of the BsAb to inhibit the proliferation of tuman cells was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit; the potential signaling pathway involved was identified by Western Blot. Cytokine secreted by PHA-L stimulated PBMC was measured by ELISA. Effects of BsAb on PBMC-mediated lysis of MKN45 cells was measured by LDH cytotoxicity assay. Results: Based on the original sequences of PD-1 and c-MET mAb, a BsAb gene was designed, cloned into pCEP4 vector for expression in 293E cells. The BsAb was obtained after purification of the cell culture supernatant. It can bind to PD-1 and c-MET simultaneously, the calculated affinity was 11.5 nM for PD-1 and 9.09 nM for c-MET. The BsAb enhanced IFN-γ production over control IgG by 2-3 folds. It also inhibit the c-MET pathway activation and the proliferation of tumor cells significantly, comparable to JnJ-38877605. The BsAb showed dose-dependent cytotoxic activity against MKN45 cells. Conclusion: Our results indicated that a novel BsAb recognizing PD-1 and c-MET was successfully generated. It could redirect T cells to kill tumor cells, while retaining its inherent ability to restore T cells and inhibit tumor cells. With this potential, this BsAb could be developed as a therapeutic candidate for the treatment of various solid tumors.

Background: Protein aggregation is a wide-ranging phenomenon. Protein aggregation mainly includes two types: one is amorphous aggregation, the other is amyloid aggregation. In particular, amyloid aggregation in vivo can cause several fatal diseases. We have investigated the influence of resveratrol on the amyloid aggregation of β-lactoglobulin. The results demonstrated resveratrol inhibited the amyloid aggregation and enhanced the amorphous aggregation of β- lactoglobulin. Objectives: The main objective of this study was to investigate the effects of resveratrol on the amyloid aggregation of β-lactoglobulin. Methods: β-lactoglobulin was incubated at pH 2.0 and 70 °C. ThT fluorescence, Congo red and transmission electron microscopy were used to monitor the formation of amyloid aggregates. In addition, resveratrol was added intoβ-lactoglobulin solutions. Intrinsic fluorescence, Circular dichroism and 1-Anilinonaphthalene-8-sulfonic Acid (ANS) fluorescence were used to investigate conformational and hydrophobic changes. Furthermore, we also studied the effect of resveratrol on the amyloid aggregation of β-lactoglobulin by using ThT fluorescence, Congo red and transmission electron microscopy at acidic pH and high temperature. Results: ThT fluorescence, Congo red and transmission electron microscopy analysis showed that β-lactoglobulin could form the amyloid fibrils when it was incubated under acidic pH and high temperature conditions. At the same time, the analysis also demonstrated resveratrol inhibited the formation of amyloid aggregates and enhanced the formation amorphous aggregates. Intrinsic fluorescence, Circular dichroism and 1-Anilinonaphthalene-8-sulfonic Acid (ANS) fluorescence analysis indicated that resveratrol could alter the conformation and increased the hydrophobicity of β- lactoglobulin. Conclusion: Our results indicated that resveratrol could effectively inhibit the formation of amyloid aggregates and enhance the formation of amorphous aggregates of β-lactoglobulin. Thus, resveratrol could be a potential inhibitor for preventing the formation of amyloid aggregates.

Background: The bursa of Fabricius (BF) is an acknowledged central immune organ, and is important to B cell differentiation. Bursal hexapeptide (BHP) is the recently reported bursalderived peptide, while its inducing function on immune response is uncertain. Objectives: The main objective of this study was to analyze the immune responses to JEV vaccine in mice induced by BHP plus JEV vaccine, and to detect the signal and biological functions of BHP on immature B cells. Methods: Mice were immunized with Japanese encephalitis virus (JEV) vaccine and BHP from 0.01 mg/mL to 0.25 mg/mL to detect antibody response and cellular immune response, respectively. The production of IgG, IgG1 and IgG2a specific to JEV in serum from immunized mice were measured by ELISA, and T cell subpopulation from immunized mice were detected with using fluorochrome conjugated mAbs of the corresponding PE-Cys/FITC/PE by flow cytometry. Spleen cells from all immunized mice were harvested after one week of second immunization for lymphocyte proliferation assay. Mouse immature B cell WEHI-231 cell was treated with 0.01μg/mL BHP for 4h, and analyzed the involved biological function and pathway of differentially expressed genes with gene microarray. Results: BHP co-immunization with JEV vaccine generated significant increased antibody levels, neutralizing antibody titers and spleen lymphocyte viability, compared to that of vaccine control. The subpopulations of T cells in spleen lymphocytes were significantly modified in the mice coimmunized with JEV vaccine and BHP. The analysis results of gene expression profiles of WEHI- 231 mouse immature B cells with BHP treatment showed that the regulated genes with BHP treatment were involved various immune related biological functions, including proliferation and activation of lymphocyte and T cell, T cell mediated immunity and regulation of adaptive immune response. Furthermore, BHP stimulated three significant enriched pathways, including amphetamine addiction, long-term potentiation, and RIG-I-like receptor signaling pathway. Conclusion: Our results indicated BHP induced significant humoral and cellular immunity to JEV vaccine, and regulated various biological processes and signalling related to immune activation in immature B cells. These results proposed the immunomodulatory function and mechanism of BHP on immune induction, which provided the novel insight on the candidate reagent for immune improvement.

Background: Amyloidosis is defined as a generic term given to a series of proteins/ polypeptides in the form of amyloid fibrils that are deposited in the tissues and give rise to a set of clinical disorders. Objectives: This work developed an approach to first examine chain association propensities of several amyloidogenic peptides: SNNFGAILSS from the islet amyloid polypeptide (coded IAPP), NAGDVAFV from the protein responsible for corneal amyloidosis (coded Lactoferrin), and (1-42) β-amyloid (coded Amyloid). Methods: Fmoc-synthesis protocol was applied for the synthesis of IAPP and Lactoferrin whereas Amyloid was synthesized through the Boc-chemistry as early detailed. Results and Conclusion: The fluorescence and light scattering experiments results indicated that Amyloid revealed a surprising reduction in the aggregation process as a function of time (decrease of about 20-30% in 3 days) through both methods. In contrast, the aggregation intensity of IAPP increased around 35% after 3 days via a light scattering procedure. These findings are very relevant for interpretation of the aggregation phenomenon of amyloidogenic peptides. The final part of this work proposed rules for dissolution of aggregated structures based on the Lewis acid and Lewis base properties of solvents. Very low solubility values (6 to 15%) were measured for peptides in water but with increased to around 90% in strong nucleophilic or strong electrophilic organic solvents. However, care should be taken when strong nucleophilic solvents such as DMSO are mixed with the strong electrophilic such as water. Both solvent molecules tend to attract each other rather than to dissolve peptide chains thus lowering the capacity of this type of solution for fibril dissolution.

Background: Wheat germ, one of the byproducts of flour milling, contains abundant physiologically active components. Globulins in wheat germ are a class of high-quality functional proteins and have received widespread attention. However, the composition of wheat germ globulin#136; WGG#137;and the structure of the typical proteins have not yet been proved. The immunological activities and immune mechanisms of the WGG have not yet been revealed in vivo. Objectives: The proteomic analysis of WGG and the structure simulation of typical proteins were studied. The immunoregulatory effects of WGG on immunosuppressed mice induced by cyclophosphamide were investigated, and the immunological activities of WGG were explored. Methods: The main components, functions, and metabolic signaling pathways of WGG were analyzed through a combination of LC-MS method and bioinformatics. The structure of WGG was predicted via the Phyre2 tool. Immunosuppression in mice was induced by cyclophosphamide. After an intraperitoneal injection of WGG for 10 days, organ indexes and pathological changes of mice were detected. The T-cell subgroups in peripheral blood were analyzed via flow cytometry. Levels of IL-2, IL-4, TNF-α, and IFN-γ were evaluated through ELISA. The mRNA expression levels of T-Bet and GATA-3 were measured using real-time PCR. Results: The results indicated that the main functional components of WGG were wheat germ globulins, histones, heat shock proteins (HSPs), and other functional proteins. Wheat germ globulins and HSPs were the major immune components of WGG. WGG significantly reduced immunosuppression in the spleen and thymus indexes (P<0.01), and mitigated the damage caused by cyclophosphamide in the spleen and thymus. Moreover, WGG significantly increased the CD4+/CD8+ of the immunosuppressed mice (P<0.01), restored Th1/Th2 imbalance (P < 0.01), enhanced the content of IL-2 and IL-4 (P<0.01), and modified the abnormal secretion of cytokines. WGG also observably reduced the mRNA expression of T-Bet and GATA-3 (P<0.01). These results manifested that WGG components improved the immune system. The action mechanisms might be related to the variation of Th1/Th2 cells resulted from the control of the mRNA expression levels of T-Bet and GATA-3. Conclusion: The wheat germ histone family and the HSPs are the major immune components of WGG. It may be the immune mechanism of WGG that these globulins affect the differentiation of Th1/Th2 cells via controlling the mRNA expression levels of related genes. The results indicated the potential application of WGG or its further purified products as a superior plant-derived immunomodulator in the future.