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);

Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma

“…Finally, it may be possible in the future to reconstitute the expression of DAPK1 in patients lacking this important tumor suppressor as delivery of its constitutive kinase domain via a CD22-specific immunoligand has shown remarkable in vitro efficacy and selectivity in preclinical testing [39]. …”

Critical Issues in the Development of Immunotoxins for Anticancer Therapy

“…92 To prevent the hypersensitivity and intrinsic immunogenicity associated with nonhuman toxins, human endogenous cytotoxic proteins have been utilized as an alternative payload in fourth-generation ITs. 21,93 Such ITs have been generated based on GrB, 94,95 angiogenin, 96 pancreatic RNase A, 8 microtubule-associated protein tau, 97 and deathassociated protein kinase 98 and have shown in vitro and in vivo antitumor activities. Nonetheless, the cytotoxicity of human enzymatic toxins is compromised by endogenous inhibitors in the cytosol: GrB is suppressed by serine protease inhibitor B9 (serpin B9 or PI9), 22 and pancreatic RNase A by the ribonuclease inhibitor (RI) protein.…”

Restoration of DAP Kinase Tumor Suppressor Function: A Therapeutic Strategy to Selectively Induce Apoptosis in Cancer Cells Using Immunokinase Fusion Proteins

“…It is also believed that DAPk2 may function upstream of DAPk1 as the overexpression of a dominant negative DAPk1 reduced DAPk2 induced cell death [ 39 ]. Notably, the downstream pathways (Caspase- and p53-dependent vs. independent apoptosis) and biologic outcomes (i.e., apoptosis vs. autophagic cell death vs. necroptosis) mediated by DAPk1 and DAPk2 are dictated in a cell specific context and nature of the upstream signal [ 47 ]. For example, a caspase dependent cell death was induced in primary fibroblast after the overexpression of DAPk1 and DAPk2.…”

“…In recent years, a better understanding of the cellular/molecular activities of the DAPk family members has allowed the design of recombinant immunokinase fusion proteins that can restore apoptosis when targeted into the cytoplasm of cancer cells. This has led to the development of DAPk1 and DAPk2 based fusion proteins for the treatment of cancer [ 47 , 63 ].…”

“…Mutants of DAPk1 lacking the CaM domain have allowed the generation of constitutively active DAPk1 [ 25 ] for immunotherapy. Using this approach, several advantages have come forward from the work of Lilienthal et al by targeting the CD22 receptor on chronic lymphocytic leukaemia (CLL) with the recombinant antibody fragment SGIII fused to a DAPk1 mutant lacking the native auto-inhibitory CaM domain [ 47 ]. Indeed, these findings clearly demonstrate that the immunokinase fusion protein (hCFP) termed DK1KD-SGIII specifically and efficiently killed CD22-positive cells of B-cell lymphoma lines (2 Burkitt’s, 1 FL/DLBCL, 2 CLL-derived) and primary CLL samples (IC 50 ranging from 275 to 875 nM), without affecting CD22-negative cells from healthy donors or CLL-patients.…”