A Novel Recombinant Anti-CD22 Immunokinase Delivers
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Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0685 Large Molecule Therapeutics Molecular Cancer Therapeutics A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death- Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells Nils Lilienthal1,2, Gregor Lohmann1, Giuliano Crispatzu1, Elena Vasyutina1, Stefan Zittrich3, Petra Mayer1, Carmen Diana Herling4, Mehmet Kemal Tur5, Michael Hallek4, Gabriele Pfitzer3, Stefan Barth6,7, and Marco Herling1,4 Abstract The serine/threonine death-associated protein kinases (DAPK) SGIII against the B-cell–exclusive endocytic glyco-receptor CD22 provide pro-death signals in response to (oncogenic) cellular stres- was created. Its high purity and large-scale recombinant production ses. Lost DAPK expression due to (epi)genetic silencing is found in a provided a stable, selectively binding, and efficiently internalizing broad spectrum of cancers. Within B-cell lymphomas, deficiency of construct with preserved robust catalytic activity. DK1KD-SGIII the prototypic family member DAPK1 represents a predisposing or specifically and efficiently killed CD22-positive cells of lymphoma early tumorigenic lesion and high-frequency promoter methylation lines and primary CLL samples, sparing healthy donor– or CLL marks more aggressive diseases. On the basis of protein studies and patient–derived non-B cells. The mode of cell death was predom- meta-analyzed gene expression profiling data, we show here that inantly PARP-mediated and caspase-dependent conventional apo- within the low-level context of B-lymphocytic DAPK, particularly ptosis as well as triggering of an autophagic program. The notori- CLL cells have lost DAPK1 expression. To target this potential ously high apoptotic threshold of CLL could be overcome by vulnerability, we conceptualized B-cell–specific cytotoxic reconsti- DK1KD-SGIII in vitro also in cases with poor prognostic features, tution of the DAPK1 tumor suppressor in the format of an immu- such as therapy resistance. The manufacturing feasibility of the novel nokinase. After rounds of selections for its most potent cytolytic CD22-targeting DAPK immunokinase and its selective antileukemic moiety and optimal ligand part, a DK1KD-SGIII fusion protein efficiency encourage intensified studies towards specific clinical containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv application. Mol Cancer Ther; 1–14. Ó2016 AACR. Introduction death-protective cluster in the presence of cytotoxic signals (1, 2). Knowledge on DAPKs has evolved since then with DAPK1 Functional screens with antisense libraries had enriched established as the prototypic serine-threonine kinase of a family death-associated protein kinases (DAPK) as a prominent of 5 proapoptotic proteins. They encompass DAPK2 (DAPK1- Related Protein 1 ¼ DRP-1), zipper-interacting protein kinase 1 Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence (ZIPK ¼ DAPK3), DAP-related apoptotic kinase (DRAK) 1, and Cluster for Cellular Stress Response and Aging-Associated Diseases – (CECAD), University of Cologne, Koln,€ Germany. 2Federal Institute for DRAK2 (1 4). They share a 50% to 80% homology of their Drugs and Devices (BfArM), Bonn, Germany. 3Institute of Vegetative catalytic domains. Loss-of-function of the DAPK tumor suppres- € 4 Physiology; University of Cologne, Koln, Germany. Department I of sors is implicated as a central tumor-initiating and metastasis Internal Medicine, Center for Integrated Oncology (CIO) Koln-Bonn,€ and CECAD, University of Cologne, Koln,€ Germany. 5Institute of -promoting event in a broad range of cancers, that is, of breast, Pathology, University Hospital, Justus Liebig University Gießen, lung, head and neck, gastrointestinal, and the hematopoetic 6 Gießen, Germany. Department of Experimental Medicine and Immu- system (1, 5, 6). The causes of tumor-associated DAPK deficiency notherapy, Institute for Applied Medical Engineering, RWTH Aachen, Aachen, Germany. 7South African Research Chair in Cancer Biotech- are primarily epigenetic mechanisms (promoter hypermethyla- nology, Institute of Infectious Disease and Molecular Medicine (IDM), tion), but also genomic deletions with subsequent LOH are Department of Integrative Biomedical Sciences, Faculty of Health observed(1,7,8). Sciences, University of Cape Town, South Africa. The DAPK proteome of multimeric complexes involves phos- Note: Supplementary data for this article are available at Molecular Cancer phorylated substrates that transmit apoptotic or autophagic sig- Therapeutics Online (http://mct.aacrjournals.org/). nals in response to cellular stresses including those by redox N. Lilienthal and G. Lohmann contributed equally to this article. burden or oncogenes (1, 2, 9). Robust protective DAPK function Corresponding Author: Marco Herling, Laboratory of Lymphocyte Signaling and promotes p19ARF-mediated p53 activity (10). Loss of DAPK Oncoproteome, Department of Internal Medicine I, University Hospital of proficiency is tumorigenic in the context of oncogenic stress, that Cologne, Building 15, Level 1, Room 1.010, Kerpener Str. 62, Cologne 50937, is, by Myc- or E2F1, as this attenuates safeguarding p53-conferred Germany. Phone: 49221-478-5194; Fax: 49221-478-88847; E-mail: cell death. Lost DAPK can also no longer retain oncogenic ERK1/ [email protected] 2 in the cytoplasm (11). The operational downstream pathways doi: 10.1158/1535-7163.MCT-15-0685 (caspase- and p53-dependent vs. -independent apoptosis; ref. 1) Ó2016 American Association for Cancer Research. and cell biologic outcomes [i.e., apoptosis (1, 10) versus www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0685 Lilienthal et al. autophagic preservation (1) vs autophagic cell death (12, 13) vs. and 2014 from the DSMZ and from the ATCC. Only original stock necroptosis (14)] appear cell context specific and to be dictated by propagated immediately upon arrival for 2 to 3 passages was the nature of the upstream stressor and the executing DAPK family picked for studies and cultures terminated after the 10th round of member (1, 2, 15). passaging (5–7 weeks). Upon thawing for experimentation Reduced DAPK1 mRNA transcription due to promoter hyper- between 2012 and 2015, all lines were authenticated by flow methylation and aberrant distribution of affected CpG islands cytometry confirming their characteristic immunophenotype, was demonstrated in virtually all cases of sporadic chronic lym- including Ig composition and TCL1 levels (28–30). The same phocytic leukemia (CLL; refs. 6, 16). In addition, a CLL haplotype applied to the FL/DLBCL lines LP, FN, CJ, LR, MS, DBr, DS, EJ with allele-specific imbalances of DAPK1 expression indicates established and provided by Dr. R.J. Ford (UT M.D. Anderson that recurring germline single-nucleotide variants and promoter Cancer Center, Houston, TX). Their identity has been described methylation can be considered as predisposing to (familial) CLL previously (29, 31). Further details are indicated in the Supple- (16, 17). DAPK1 promoter hypermethylation was also shown to mentary Data. Experimentations were done in suspension cul- be associated with more aggressive disease and adverse clinical tures under conditions as instructed and published (28, 29). outcome in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL; refs. 5, 18). Western blots, IHC, and flow cytometry Giventheircentralroleintumorigenesisandclonalsuste- Protocols and reagents for immunoblots (DAPKs and apopto- nance, DAPKs have been intensely investigated as therapeutic tic proteins), IHC (DAPK1), and flow cytometry are outlined in targets. Strategies employ gene therapy, development of func- the Supplementary Data. tional DAPK activators, or demethylating agents to reestablish DAPK expression (19, 20). This also extents (reversely) to In silico meta-analysis of DAPK family gene expression from DAPK inhibitors in autoinflammatory disease and graft rejec- array-based data tion (15, 20–22). We explored here if reinstating DAPK activity Via manual search of literature and data repositories, we would perturb the high apoptotic threshold in DAKP-deficient obtained available primary data out of 8 reports on array-based neoplastic B cells that are burdened by incurring (oncogenic) gene expression profiling (GEP) of CLL samples with specific stress. We further hypothesized that an efficient immunoli- controls. Details and further meta-analyses on B-cell subsets and gand-based targeting would potentiate that selectivity. Our other B-cell lymphomas are found in the Supplementary Data. All DAPK20-CD30L immunokinase already showed encouraging datasets were separately background corrected and annotated in vitro (23) and in vivo (24) activity in systems of Hodgkin using the BioConductor packages "affy" and "biomaRt" in lymphoma. R-3.1.0. The datasets were quantile-normalized and replicates A novel CD22-targeting construct containing a constitutively were combined by mean. Sample names were assigned from the active DAPK1 variant showed here efficient binding, internaliza- GEO (32) entry to test for differential expression via t test or tion, and cell death induction in malignant B cells. This immu- Wilcoxon/Mann–Whitney test. Only the highest fold change nokinase revealed a favorable in vitro profile of induced apoptosis (FC; P < 0.1) was obtained and visualized in the summarizing and autophagy,