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[PMC free article] [PubMed] [Google Scholar] 13. blood mononuclear cells (PBMC) and purified NK cells. Importantly, TP15-Fc showed potent efficacy and completely prevented growth of human INA-6.Tu1 plasma cells in a xenograft SCID/beige mouse model. Thus, the novel ADCC-optimized TP15-Fc exerts potent anti-myeloma activity and has promising characteristics to be further evaluated for MM immunotherapy. [27]. In addition, anti-myeloma brokers that impair interactions between the bone marrow (BM) microenvironment and malignant plasma cells can be of particular interest [28]. Cell surface proteins which are involved in myeloma cell adhesion to BM stromal cells (BMSC) could be potential targets for therapeutic mAbs. Those include members of the integrin and adhesion protein families and their natural receptors, e.g. vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1/CD54). Increased serum levels of both, VCAM-1 and ICAM-1, were reported to be associated with advanced disease and poor outcome in MM patients [29]. To identify antibodies targeting cell surface antigens on malignant plasma cells that have potential as immunotherapeutic brokers, we have employed phage display technology with human Rodatristat single chain fragment variable (scFv) antibody libraries and a cellular panning strategy. Phage PIII-15 was selected based on its favorable binding profile and converted into a human scFv-Fc fusion protein named TP15-Fc, Rodatristat that specifically targets human ICAM-1/CD54. TP15-Fc induced significant ADCC against myeloma cells and, importantly, completely prevented MM growth supernatants containing single phage antibodies tested with JK-6L and CEM cells in ELISA showed strong and unique reactivity with the JK-6L MM cells. Hence, the applied panning strategy resulted in the successful isolation of monoclonal phage antibodies binding to myeloma cell lines. Of note, since a fixed volume (100 l) of phage-containing supernatants without prior quantification were used in this ELISA experiment, no direct comparison between the binding properties of the single phage antibodies can be made. By screening defined quantities of 51010 single phage clones from panning rounds 2 and 3, phage PIII-15, obtained from the third round of panning, was selected for further functional analyses due to its significant binding to different myeloma/plasma cell leukemia (PCL) and Burkitt’s lymphoma cell lines, while binding to other leukemia cell lines (CEM, KG-1a), PBMC and the indicated CAGH1A leukocyte subpopulations was not observed (Physique ?(Figure1D).1D). Importantly, PIII-15 also bound to CD138+ malignant plasma cells of a PCL patient, whereas no reactivity was Rodatristat observed with CD3+ T lymphocytes and CD56+ cells Rodatristat (predominantly NK cells) of an healthy individual (Physique ?(Figure1E1E). Open in a separate window Physique 1 Binding characteristics of phages after panningAll cellular ELISA and flow cytometry experiments were performed with 0.5106 cells per sample. Bound phages were either detected with a FITC-labeled anti-fd bacteriophage antibody (flow cytometry) or with an HRP-labeled anti-M13 antibody (ELISA). (A) 2.51011 phages from the original (input) or the panned libraries from round 1 to 3 (Panning I to III) were incubated with the indicated cells and binding was tested in whole-cell ELISA. Mean values SEM from duplicates are given. (B) Flow cytometric analyses of phages from Tomlinson I (left panel) and J library (right panel) prior to panning (black lines) and from Rodatristat panning rounds 1 (light red and blue line, respectively), 2 (dark red and blue line, respectively), and 3 (grey lines) with myeloma (INA-6 and JK-6L) and leukemia cell lines (CEM and KG-1a) are shown. (C) Binding of monoclonal phage antibody-containing TG1 supernatants (100 l each) from Tomlinson I library, panning round 3, was tested in ELISA experiments with JK-6L and CEM cells. (D) Binding characteristics of the monoclonal phage antibody PIII-15 (51010 phages) were evaluated by whole-cell ELISA. Mean values SEM from three impartial experiments with duplicates are given. (***) p 0.001 mean absorbance of PIII-15 ctrl phage. (E) Flow cytometric experiments were performed using INA-6, primary patient cells (7-AAD-/CD138+; frozen sample from a plasma cell leukemia patient with 86 % malignant plasma cells) and PBMC subsets of a healthy donor (7-AAD- and.