Yuanfu Xu

Efficient inhibition of human B-cell lymphoma xenografts with an anti-CD20×anti-CD3 bispecific diabody

Bispecific antibodies have been exploited both as cancer immunodiagnostics and as cancer therapeutics, and have shown promise in several clinical trials in cancer imaging and therapy. A number of bispecific antibodies against B-cell markers have been shown to be effective in vitro in mediating tumor cell lysis and in vivo in inhibiting tumor growth in animal models. We have constructed a bispecific diabody from the variable genes encoding two hybridoma-derived monoclonal antibodies directed against human CD20 on B cells and CD3 on T cells. The anti-CD20 x anti-CD3 diabody was expressed in a single Escherichia coli host and purified by a one-step affinity chromatography. The bispecific diabody bound as efficiently to both CD20- and CD3-positive cells as the respective parental antibodies, and was capable of cross-linking CD20-positive tumor cells and human T lymphocytes as shown by cellular rosetting. The diabody effectively lysed human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Further, when combined with human PBL and interleukin-2, the diabody significantly prolonged the survival of nude mice inoculated with human B-lymphoma cells. Taken together, our results suggest that an anti-CD20 x anti-CD3 diabody may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.

Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux.

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37°C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.

Extracellular Acidification Acts as a Key Modulator of Neutrophil Apoptosis and Functions.

In human pathological conditions, the acidification of local environment is a frequent feature, such as tumor and inflammation. As the pH of microenvironment alters, the functions of immune cells are about to change. It makes the extracellular acidification a key modulator of innate immunity. Here we detected the impact of extracellular acidification on neutrophil apoptosis and functions, including cell death, respiratory burst, migration and phagocytosis. As a result, we found that under the acid environment, neutrophil apoptosis delayed, respiratory burst inhibited, polarization augmented, chemotaxis differed, endocytosis enhanced and bacteria killing suppressed. These findings suggested that extracellular acidification acts as a key regulator of neutrophil apoptosis and functions.

Positive Regulation of Interleukin-1β Bioactivity by Physiological ROS-Mediated Cysteine S-Glutathionylation

Reactive oxygen species (ROS)-induced cysteine S-glutathionylation is an important posttranslational modification (PTM) that controls a wide range of intracellular protein activities. However, whether physiological ROS can modulate the function of extracellular components via S-glutathionylation isxa0unknown. Using a screening approach, we identified ROS-mediated cysteine S-glutathionylation on several extracellular cytokines. Glutathionylation of the highly conserved Cys-188 in IL-1β positively regulates its bioactivity by preventing its ROS-induced irreversible oxidation, including sulfinic acid and sulfonic acid formation. We show this mechanism protects IL-1β from deactivation by ROS in an inxa0vivo system of irradiation-induced bone marrow (BM) injury. Glutaredoxin 1 (Grx1), an enzyme that catalyzes deglutathionylation, was present and active in the extracellular space in serum and the BM, physiologically regulating IL-1β glutathionylation and bioactivity. Collectively, we identify cysteine S-glutathionylation as a cytokine regulatory mechanism that could be a therapeutic target in the treatment of various infectious and inflammatory diseases.

Integrated Biophysical and Biochemical Signals Augment Megakaryopoiesis and Thrombopoiesis in a Three-Dimensional Rotary Culture System

Platelet transfusion has been widely used in patients undergoing chemotherapy or radiotherapy; however, the shortage of the platelet supply limits the care of patients. Although derivation of clinical‐scale platelets in vitro could provide a new source for transfusion, the devices and procedures for deriving scalable platelets for clinical applications have not been established. In the present study, we found that a rotary cell culture system (RCCS) can potentiate megakaryopoiesis and significantly improve the efficiency of platelet generation. When used with chemical compounds and growth factors identified via small‐scale screening, the RCCS improved platelet generation efficiency by as much as ∼3.7‐fold compared with static conditions. Shear force, simulated microgravity, and better diffusion of nutrients and oxygen from the RCCS, altogether, might account for the improved efficient platelet generation. The cost‐effective and highly controllable strategy and methodology represent an important step toward large‐scale platelet production for future biomedical and clinical applications.

MEIS1 Regulates Hemogenic Endothelial Generation, Megakaryopoiesis, and Thrombopoiesis in Human Pluripotent Stem Cells by Targeting TAL1 and FLI1

Summary Human pluripotent stem cells (hPSCs) provide an unlimited source for generating various kinds of functional blood cells. However, efficient strategies for generating large-scale functional blood cells from hPSCs are still lacking, and the mechanism underlying human hematopoiesis remains largely unknown. In this study, we identified myeloid ectopic viral integration site 1 homolog (MEIS1) as a crucial regulator of hPSC early hematopoietic differentiation. MEIS1 is vital for specification of APLNR+ mesoderm progenitors to functional hemogenic endothelial progenitors (HEPs), thereby controlling formation of hematopoietic progenitor cells (HPCs). TAL1 mediates the function of MEIS1 in HEP specification. In addition, MEIS1 is vital for megakaryopoiesis and thrombopoiesis from hPSCs. Mechanistically, FLI1 acts as a downstream gene necessary for the function of MEIS1 during megakaryopoiesis. Thus, MEIS1 controls human hematopoiesis in a stage-specific manner and can be potentially manipulated for large-scale generation of HPCs or platelets from hPSCs for therapeutic applications in regenerative medicine.

Efficacy of anti-CD20 chimeric Fab’ fragment on proliferation of B lymphoma cells

The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HI47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed inE. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI47 to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’ fragment inhibited the proliferation of Daudi cells, IC50 = 69 μg/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, Ka was about 8.9×108 (mol/L)−1.

MSX2 Initiates and Accelerates Mesenchymal Stem/Stromal Cell Specification of hPSCs by Regulating TWIST1 and PRAME

Summary The gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Correction: Extracellular Acidification Acts as a Key Modulator of Neutrophil Apoptosis and Functions

The Funding section is incorrect. The correct funding information is as follows: This study was supported by grants from the National Basic Research Program of China (2012CB966403 and 2015CB964903), the Chinese National Natural Science Foundation (31271484, 31471116, and 31171431), and the Tianjin Natural Science Foundation (12JCZDJC24600).