Abstract
Ranpirnase (Rap) is an amphibian ribonuclease showing anti-tumor activity in clinical studies. We have previously reported that targeted delivery of Rap by chemical conjugation or recombinant fusion with antibodies specific for CD22, CD74 and Trop-2 could enhance its in vitro cytotoxicity as high as 10,000-fold in selected malignant cell lines. The DOCK-AND-LOCKTM (DNLTM) platform technology is a powerful method to construct novel agents of defined composition and retained bioactivity by site-specific conjugation of two types of modules, one containing the dimerization and docking domain (DDD) of cAMP-dependent protein kinase A (PKA), referred to as the DDD module, and the other bearing the anchoring domain (AD) of an interactive A-kinase anchoring protein (AKAP), referred to as the AD module. Among the distinctive features of DNL are the spontaneous formation of a dimer of the DDD module and the self-assembly of the DDD module with the AD module into a non-covalent complex, which is subsequently rendered covalent with disulfide bonds to enhance stability in vivo. The amino acid sequences of a pair of DDD and AD linkers useful for the DNL conjugation are termed DDD2 and AD2, respectively. To further explore the potential of Rap-based immunotoxins, we expressed a DDD2-module of Rap in E. coli and linked the resulting dimer of Rap to an AD2-module of a humanized IgG (expressed in myeloma cells) at each of the carboxyl termini of either the light chain (the CK-format) or the heavy chain (the CH3-format), thus producing a class of novel immunoRNases with quadruple Rap. To date, we have evaluated a pair of such constructs, 22* -Rap and 22-Rap, comprising four copies of Rap linked to the CK or CH3 termini of epratuzumab (humanized anti-CD22), respectively, in a panel of CD22-expressing human lymphoma/leukemia cell lines, which include Burkitt lymphoma (Daudi, Raji, Ramos), acute lymphoblastic leukemia (REH, 697, and RS4;11), and mantle cell lymphoma (Granta-519 and Jeko-1). The results of the MTS assay indicated that 22* -Rap was highly active (EC50 ≤ 1 nM) against Daudi, Ramos, Raji, REH, 697, RS4;11, and Granta-519 cells. Comparable cytotoxicity with EC50 values in the subnanomolar range also was observed for 22-Rap in Ramos, Daudi and Jeko-1 cells. In contrast, neither the individual DNL components (IgG-AD2 or Rap-DDD2), tested alone or in combination, nor E1* -Rap, the counterpart of 22* -Rap, generated by substituting epratuzumab with hRS7 IgG (humanized anti-Trop-2), showed notable cytotoxicity. In a disseminated Daudi xenograft model in which treatment with 10 or 20 µg of 22* -Rap (q4dx4) started 7 days after intravenous inoculation of the tumor cells, all 10 mice (5 in each group) survived over 126 days and were tumor-free, whereas the control groups, treated with saline, epratuzumab (25 µg, q4dx4), or the same dose-schedule of nonspecific control, E1* -Rap, succumbed within 36 days. These promising results, together with the findings that 22* -Rap at 10 nM was marginally toxic to B cells and other hematological cells in PBMCs, encourage further development of 22* -Rap or 22-Rap for therapy of CD22-expressing lymphomas and leukemias.
Disclosures:
Liu: IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc.: Employment, Stock option Other. Cardillo:Immunomedics, Inc.: Employment, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc: Employment, Stock option Other.
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