ABSTRACT
BACKGROUND: Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse, and express the well-known surface marker CD133. METHODS: DNA fragments from two fully humanized single chain fragment variable (scFv) antibodies recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigens to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated a unique antibody that recognized the molecular framework and was species cross-reactive. RESULTS: In vitro chromium-51 ((51)Cr) release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5 % CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. CONCLUSION: These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133 and that this anti-CD133 scFv may be useful in this bispecific platform or perhaps in the design of more complex trispecific molecules for carcinoma therapy.
Subject(s)
AC133 Antigen/immunology , Antigens, Neoplasm/immunology , Colorectal Neoplasms/immunology , Killer Cells, Natural/immunology , Single-Chain Antibodies/immunology , Animals , Humans , MiceABSTRACT
BACKGROUND: Upon coincubation with platelet aggregates, CD34(+) progenitor cells have the potential to differentiate into foam cells. There is evidence that progenitor cells from diabetic and nondiabetic patients have different properties, which may affect the patients' prognosis. In this study we investigated an in vitro model of foam cell formation based on patient-derived CD34(+) progenitor cells. We analyzed the growth characteristics as well as the M-CSF-release and matrix metalloproteinase (MMP) synthesis from CD34(+) progenitor cell-derived foam cells originating from diabetic and nondiabetic patients. METHODS AND RESULTS: Bone marrow samples were obtained from 38 patients who were elected for thoracic surgery. CD34(+) progenitor cells from diabetic and nondiabetic patients were isolated and incubated with platelets from healthy volunteers. Foam cell formation was confirmed by immunostaining (CD68) and quantified by light microscopy. Whereas the absolute number of foam cells was not affected, the negative slope in the growth curve was seen significantly later in the diabetic group. In supernatants derived from"diabetic" CD34(+) progenitor cells, MMP-9 was significantly enhanced, whereas MMP-2 activity or M-CSF-release was not affected significantly. CONCLUSION: In a coculture model of CD34(+) progenitor cells with platelets, we show for the first time that"diabetic" CD34(+) progenitor cells exhibit functional differences in their differentiation to foam cells concerning growth characteristics and release of MMP-9.
Subject(s)
Diabetes Mellitus/enzymology , Diabetes Mellitus/pathology , Foam Cells/enzymology , Foam Cells/pathology , Matrix Metalloproteinase 9/metabolism , Mesenchymal Stem Cells/pathology , Aged , Antigens, CD34/metabolism , Blood Platelets/enzymology , Blood Platelets/pathology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Coculture Techniques , Enzyme Activation , Female , Humans , Male , Mesenchymal Stem Cells/enzymologyABSTRACT
Natural killer (NK) cells play an important role in the immunosurveillance of hematopoietic malignancies. Their reactivity is influenced by activating and inhibitory signals mediated by tumor-expressed ligands for NK receptors. Many members of the tumor necrosis factor (TNF) family modulate differentiation, proliferation, activation and death of both tumor and immune effector cells. The TNF receptor family member glucocorticoid-induced TNFR-related protein (GITR) stimulates anti-tumor immunity in mice, but available data indicate that GITR may mediate different effects in mice and men and impairs the reactivity of human NK cells. Here, we comprehensively studied the expression and function of GITR ligand (GITRL) in leukemia. Among the different leukemia entities, pronounced expression of GITRL on leukemic cells was observed in chronic lymphocytic leukemia (CLL), and the GITR receptor was expressed at significantly higher levels on NK cells of CLL patients compared with healthy controls. Upon GITR-GITRL interaction, signaling via GITRL into the leukemia cells induced the release of interleukin (IL)-6, IL-8 and TNF, which act as growth and survival factors for CLL cells. In addition, GITRL impaired both direct and Rituximab-induced degranulation, cytotoxicity and interferon-γ production of NK cells, which could be restored by GITR blocking antibodies. Thus, GITRL may contribute to disease pathophysiology and resistance to direct and Rituximab-induced NK reactivity in CLL.
