Ranpirnase (Rap) is a cytotoxic ribonuclease (RNase) isolated from frog oocytes. Here we describe high antitumor activity of a novel immunotoxin, 2L-Rap-hLL1-␥4P, composed of 2 Rap molecules, each fused to the N terminus of the light chain of hLL1, an internalizing anti-CD74 humanized antibody. To reduce unwanted side effects, the constant region of hLL1 was changed from ␥1 to ␥4 and further to ␥4P by replacing serine 228 to proline to prevent the formation of a half immunoglobulin G (IgG) common for IgG 4 . In vitro, 2L-Rap-hLL1-␥4P retained RNase activity, specific binding to CD74, and was significantly more potent against CD74 ؉ cell lines (Daudi, Raji, and MC/CAR) than naked hLL1. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1-␥4P was similar to that of naked hLL1. The maximum tolerated dose of 2L-Rap-hLL1-␥4P in severe combined immunodeficient mice (SCID) or BALB/c mice was 50 g per mouse. In Raji and Daudi Burkitt lymphoma xenograft models, treatment with a single 5 to 50 g dose of 2L-Rap-hLL1-␥4P, given as early or delayed treatment, resulted in cures of most animals. Treatment with 2L-Rap-hLL1-␥4P was significantly better than all controls, including saline, naked hLL1, and nonspecific immunotoxin. IntroductionRanpirnase (Rap), a monomeric protein (Mr, 11 817; 104 amino acids), is an amphibian ribonuclease (RNase) belonging to the RNase A superfamily. 1 Native Rap, isolated from Rana pipiens eggs, 2 demonstrated significant cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro 3 and in vivo. 4 Rap has a low affinity (more than 1 M) for RNase inhibitor (RI), which constitutes about 0.01% of the cytosolic protein in mammalian cells, 1 and therefore could evade inactivation by RI. As indicated by its resistance to both protease degradation and denaturation at elevated temperatures, 5 Rap is highly stable. Its enzymatic activity requires an N-terminal pyroglutamyl residue. 6 Recombinant Rap (rRap) expressed in Escherichia coli with methionine (Met) at the N terminus (Met [Ϫ1]) displays much reduced activity, 7 but glycosylation of rRap (expressed in Pichia pastoris) increases its conformational stability and toxicity to cancer cells. 8 Rap enters cells via receptor-mediated endocytosis. 9 Once internalized into the cytosol, it selectively degrades tRNA, 10,11 resulting in inhibition of protein synthesis and induction of apoptosis. 10 Immunotoxins consisting of Rap and LL2, an internalizing anti-CD22 murine monoclonal antibody (mAb), 12 have been prepared by chemical conjugation and shown to have potent and specific antitumor effects against CD22 ϩ cells both in vitro and in vivo. 13 Rap was studied in the treatment of patients with unresectable malignant mesothelioma, 14 where the reversible dose-limiting toxicity was renal and where there appeared to be no immunogenicity. 15 These encouraging results prompted us to develop a series of novel recombinant immunotoxins involving Rap and other internalizing mAbs. 16,17 CD74 (invariant chain, Ii) is a type II transmembrane glycoprotein...

No abstract

Rap, an amphibian ribonuclease, is a single-chain protein of 104 amino acids that kills cells by degrading t-RNA upon internalization. CD74 is a rapidly internalizing type-II transmembrane chaperone molecule associated with HLA-DR, and has high expression on hematological malignancies including B-cell non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM). We have constructed and evaluated two novel immunotoxins, 2L-Rap-hLL1-γ 4P and 2L-Rap(N69Q)-hLL1-γ 4P, each composed of two Rap molecules fused to hLL1, an internalizing anti-CD74 humanized monoclonal antibody. The Rap gene was inserted at the N-terminus of the light chain in the expression vector of hLL1 and expressed in NS0 mouse myeloma cells. To reduce unwanted cytotoxicity, the CH1, CH2, CH3 and the hinge regions of the γ 1 chain of hLL1 were replaced with those of γ 4. Additionally, the serine residue in the hinge region was converted to proline to prevent the formation of IgG4 half-molecules. Noting that Rap contains a potential N-glycosylation site at the 69th residue of asparagine(N69), a variant of Rap, referred to as Rap(N69Q), was constructed by changing N to Q (glutamine) and this variant was used to make 2L-Rap(N69Q)-hLL1-γ 4P. Purified recombinant immunotoxins were shown to be a single peak by SE-HPLC and their MW determined by MALDI-TOF to be 177,150, which is in agreement with the MW of one IgG (150,000) plus two Rap molecules (24,000). In vitro, both immunotoxins retained RNase activity, specific binding to CD74, and were significantly more potent against CD74-positive NHL and MM cell lines (Daudi, Raji and MC/CAR) than naked hLL1 or non-specific control immunotoxin, 2L-Rap(N69Q)-hRS7(immunotoxin against EGP-1). In Raji and Daudi Burkitt’s lymphoma xenograft models, treatment with a single 5- to 50-μg dose of 2L-Rap-hLL1-γ 4P, given as early or delayed treatment, resulted in cures of most animals. Additionally, treatment with a single 15-μg dose of 2L-Rap(N69Q)-hLL1-γ 4P 1-day post injection of cells resulted in 100% cures. Treatment with 2L-Rap-hLL1-γ 4P or 2L-Rap(N69Q)-hLL1-γ 4P was significantly better than all controls, including saline, naked hLL1 and non-specific immunotoxin. The maximum tolerated dose of 2L-Rap-hLL1-γ 4P or 2L-Rap(N69Q)-hLL1-γ 4P in SCID or BALB/c mice was 50 μg/mouse and the dose-limiting toxicity was hepatic. In our preliminary studies, we have observed that treating animals with NSAID’s, such as indomethacin, can ameliorate the hepatoxicity of 2L-Rap-hLL1-γ 4P. All animals that were injected with 100 μg/mouse 2L-Rap-hLL1-γ 4P alone died with a median survival time of 7 days; however, animals treated with 1.25mg/kg indomethacin prior and post-treatment of 2L-Rap-hLL1-γ 4P survived the duration of study (day 40). Experiments to determine the possible causes of liver toxicity produced by 2L-Rap-hLL1-γ 4P and to determine the MTD of Rap-immunotoxins in mice after treatment with indomethacin are ongoing. In conclusion, we have constructed two CD74-targeted novel recombinant immunotoxins containing Rap molecules that have demonstrated curative therapeutic effects in animal models of human B-cell lymphoma, and thus could be potential therapeutics for CD74-postive lymphomas and myelomas.

Ranpirnase (Rap), isolated from frog (Rana pipiens) oocytes, is a monomeric ribonuclease (MW 11800) that kills cells by degrading t-RNA upon internalization. Previous studies indicated that the cytotoxicity of Rap could be enhanced more than 10,000-fold when the enzyme is chemically conjugated to an internalizing antibody. Here we describe the construction and characterization of 2L-Rap-hLL1-γ4P, composed of two Rap molecules fused to hLL1, an internalizing anti-CD74 humanized monoclonal antibody. To reduce unwanted cytotoxicity, the IgG1 constant region of hLL1 was replaced with an IgG4 that contains a proline mutation in the hinge region. The Rap gene was inserted at the N-terminus of the light chain in the expression vector of hLL1 and expressed in NS0 mouse myeloma cells. The fusion protein was characterized by a variety of techniques, including SE-HPLC, SDS-PAGE, in vitro transcription translation (IVTT) assay using luciferase reporter system, and competition ELISA to measure the binding affinity for CD74. The in vitro potency was determined in non-Hodgkin’s lymphoma (Daudi) and multiple myeloma (MC/CAR) cell lines by MTS tetrazolium dye reduction assay. In vivo pharmacokinetics and biodistribution of radiolabeled 2L-Rap-hLL1- γ4P was compared to radiolabeled hLL1 mAb in naïve mice and in vivo therapeutic efficacy of 2L-Rap-hLL1- γ4P was determined in a xenograft model of Burkitt’s non-Hodgkin’s lymphoma (Daudi). Purified 2L-Rap-hLL1- γ4P was shown to be a single peak by SE-HPLC and its MW determined by MALDI-TOF to be 177,150, which is in agreement with the MW of one IgG (150,000) plus two Rap molecules (24,000). Reducing-SDS-PAGE of 2L-Rap-hLL1- γ4P revealed the presence of 3 bands, one corresponding to the heavy chain and the other two appearing to be derived from the Rap-fused light chains (38,526 and 36,700 by MS). Occurrence of the 2 light chains was shown to be due to glycosylation of Rap at the N69 residue. The binding affinity of 2L-Rap-hLL1- γ4P for CD74 was indistinguishable from that of hLL1. Both 2L-Rap-hLL1- γ4P and hLL1 bound to CD74 with subnanomolar affinity. The EC50 of RNase activity, as measured by the IVTT assay, was 300 pM for 2L-Rap-hLL1- γ4P and 30 pM for recombinant Rap (expressed in E. coil). In in vitro cytotoxicity assays, 2L-Rap-hLL1- γ4P was significantly cytotoxic against Daudi (EC50 280 pM) and the myeloma cell line, MC/CAR (EC50 50 nM). In contrast, free Rap or naked hLL1 did not demonstrate significant cytotoxicity at the concentrations tested. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1- γ4P was almost identical to that of naked hLL1. Both 2L-Rap-hLL1- γ4P and hLL1 showed biphasic clearance from the circulation; the α and β half-life (t1/2) of 2L-Rap-hLL1- γ4P were 5 h and 119 h, respectively, and those of hLL1 were 4 h and 125 h, respectively. In tissue biodistribution studies, no significant difference was observed between 2L-Rap-hLL1- γ4P and hLL1 with regards to normal tissue uptake. Early efficacy results in the Daudi Burkitt’s non-Hodgkin’s lymphoma xenograft model demonstrate that treatment with a single dose of 2L-Rap-hLL1- γ4P as low as 1 μg/mouse significantly improves survival in comparison to untreated control mice (P<0.0001).