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Tohidkia M.R.,Tabriz University of Medical Sciences | Tohidkia M.R.,Tehran University of Medical Sciences | Tohidkia M.R.,University of Tehran | Asadi F.,Tehran University of Medical Sciences | And 6 more authors.
BioDrugs | Year: 2013

Background and Objective: Gastric/gastrointestinal cancers are associated with high mortality worldwide. G-protein coupled receptor (GPCR) superfamily members such as gastrin/cholecystokinin-B receptor (CCK-BR) are involved in progression of gastric tumors, thus CCK-BR is considered as a potential target for immunotherapy. However, production of functional monoclonal antibodies (mAbs) against GPCR seems to be very challenging, in part due to its integration in cell membranes and inaccessibility for selection. To tackle this problem, we implemented phage display technology and a solution-phase biopanning (SPB) scheme for production of mAbs specific to the native conformation of CCK-BR. Methods: To perform the SPB process, we utilized a synthetic biotinylated peptide corresponding to the second extracellular loop (ECL2) of CCK-BR and a semi-synthetic phage antibody library. After enzyme-linked immunosorbent assay (ELISA) screening, the CCK-BR specificity of the selected single-chain variable fragments (scFvs) were further examined using immunoblotting, whole-cell ELISA, and flow cytometry assays. Results: After performing four rounds of selection, we identified nine antibody clones which showed positive reactivity with the CCK-BR peptide in an ELISA assay. Of these, eight clones were unique scFv antibodies and one was a VL single domain antibody. Specificity analysis of the selected scFvs revealed that five of the selected scFvs recognized a denatured form of CCK-BR, while the majority of the selected scFvs were able to recognize the native conformation of CCK-BR on the surface of human gastric adenocarcinoma cells and cervical carcinoma HeLa cells. Conclusion: For the first time, we report on the establishment of a diverse panel of scFv antibody fragments that are specific to the native conformation of CCK-BR. Based on these results, we suggest the selected scFv antibody fragments as potential agents for diagnosis, imaging, targeting, and/or immunotherapy of cancers that overexpress CCK-BR. © 2013 Springer International Publishing Switzerland. Source

Ye Q.,Ovarian Cancer Research Center | Song D.-G.,Ovarian Cancer Research Center | Poussin M.,Ovarian Cancer Research Center | Yamamoto T.,Ovarian Cancer Research Center | And 5 more authors.
Clinical Cancer Research | Year: 2014

Purpose: Upregulation of CD137 (4-1BB) on recently activated CD8+ T cells has been used to identify rare viral or tumor antigen-specific T cells from peripheral blood. Here, we evaluated the immunobiology of CD137 in human cancer and the utility of a CD137-positive separation methodology for the identification and enrichment of fresh tumor-reactive tumor-infiltrating lymphocytes (TIL) or tumor-associated lymphocytes (TAL) from ascites for use in adoptive immunotherapy. Experimental Design: TILs from resected ovarian cancer or melanoma were measured for surface CD137 expression directly or after overnight incubation in the presence of tumor cells and homeostatic cytokines. CD137pos TILs were sorted and evaluated for antitumor activity in vitro and in vivo. Results: Fresh ovarian TILs and TALs naturally expressed higher levels of CD137 than circulating T cells. An HLA-dependent increase in CD137 expression was observed following incubation of fresh enzymedigested tumor or ascites in IL-7 and IL-15 cytokines, but not IL-2. Enriched CD137pos TILs, but not PD-1pos or PD-1neg CD137neg cells, possessed autologous tumor reactivity in vitro and in vivo. In melanoma studies, all MART-1-specific CD8+ TILs upregulated CD137 expression after incubation with HLA-matched, MARTexpressing cancer cells and antigen-specific effector function was restricted to the CD137pos subset in vitro. CD137pos TILs also mediated superior antitumor effects in vivo, compared with CD137neg TILs. Conclusions: Our findings reveal a role for the TNFR-family member CD137 in the immunobiology of human cancer where it is preferentially expressed on tumor-reactive subset of TILs, thus rationalizing its agonistic engagement in vivo and its use in TIL selection for adoptive immunotherapy trials. Clin Cancer Res; 20(1); 44-55. © 2013 AACR. Source

Lynn R.C.,Ovarian Cancer Research Center | Poussin M.,Ovarian Cancer Research Center | Kalota A.,University of Pennsylvania | Feng Y.,U.S. National Cancer Institute | And 4 more authors.
Blood | Year: 2011

T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ+ AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ+ THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34+ HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, whichmay be augmented by combination with ATRA. Copyright © 2011 by The American Society of Hematology; all rights reserved. Source

Ye Q.,Ovarian Cancer Research Center | Song D.-G.,Ovarian Cancer Research Center | Powell Jr. D.J.,Ovarian Cancer Research Center
OncoImmunology | Year: 2013

We have recently identified tumor necrosis factor receptor superfamily, member 9 (TNFRS F9, best known as CD137 or 4-1BB) as a biomarker of tumor-reactive T cells naturally occurring in cancer patients, and developed a rapid, accurate system to comprehensively isolate lymphocytes with tumor-rejecting properties from human biopsies. Our findings reveal a previously unappreciated role for CD137, a co-stimulatory TNFR family member, in the immunobiology of human cancer. © 2013 Landes Bioscience. Source

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