title: A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.
abstract: The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR.
authors.affiliation: Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Köln, Germany. Federal Institute for Drugs and Devices (BfArM), Bonn, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Lilienthal
authors.affiliation: Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Köln, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Lohmann
authors.affiliation: Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Köln, Germany.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Crispatzu
authors.affiliation: Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Köln, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Vasyutina
authors.affiliation: Institute of Vegetative Physiology; University of Cologne, Köln, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zittrich
authors.affiliation: Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Köln, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Mayer
authors.affiliation: Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, and CECAD, University of Cologne, Köln, Germany.
authors.forename: Carmen Diana
authors.initials: CD
authors.lastname: Herling
authors.affiliation: Institute of Pathology, University Hospital, Justus Liebig University Gießen, Gießen, Germany.
authors.forename: Mehmet Kemal
authors.initials: MK
authors.lastname: Tur
authors.affiliation: Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, and CECAD, University of Cologne, Köln, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.affiliation: Institute of Vegetative Physiology; University of Cologne, Köln, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pfitzer
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, RWTH Aachen, Aachen, Germany. South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine (IDM), Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, South Africa.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.affiliation: Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Köln, Germany. Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, and CECAD, University of Cologne, Köln, Germany. marco.herling@uk-koeln.de.
authors.forename: Marco
authors.initials: M
authors.lastname: Herling
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D064550:Death-Associated Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005810:Multigene Family; D009154:Mutation; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000037:antagonists & inhibitors*; D057127:Single-Chain Antibodies / Q000008:administration & dosage
chemical_list: D000970:Antineoplastic Agents; D011993:Recombinant Fusion Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; D057127:Single-Chain Antibodies; D064550:Death-Associated Protein Kinases