Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin

Abstract

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...

CD74: A New Candidate Target for the Immunotherapy of B-Cell Neoplasms

“…In vivo, the pharmacokinetic profile of 2L-RaphLL1-g4P was similar to that of hLL1. Most importantly, marked therapeutic efficacy was shown in animal models of (62). Figure 6 summarizes a study in which Rap-hLL1 specifically targeted Daudi xenografts and yielded long-term survival in all treated mice.…”

Ranpirnase (Frog RNase) Targeted with a Humanized, Internalizing, Anti–Trop-2 Antibody Has Potent Cytotoxicity against Diverse Epithelial Cancer Cells

“…The construction of the plasmid pdHL2-Rap-L-hLL1-γ4P for expressing 2L-Rap-hLL1-γ4P has been described in detail (9). The expression vector pdHL2-Rap (Q)-LhLL1-γ4P was derived from pdHL2-Rap-L-hLL1-γ4P by replacing Rap with Rap(Q), and the plasmid pdHL2-Rap (Q)-L-hRS7-γ1 for expressing (Q)-hRS7 was constructed by subcloning Rap(Q) gene from pdHL2-Rap(Q)-L-hLL1-γ4P into pdHL2-hRS7-γ1 vector.…”

“…We have previously reported that 2L-Rap-hLL-γ4P retained the binding affinity and specificity for CD74 when compared with unconjugated hLL1, displayed RNase activity comparable with free Rap, showed potent in vitro cytotoxicity against CD74-positive (Daudi, Raji, and MC/CAR), but not CD74-negative (DMS 53), cell lines, and showed an excellent therapeutic index in vivo in two xenograft models of non-Hodgkin lymphoma (9). In this work, we provide a further example to illustrate that the NH 2 -terminal fusion of Rap(Q) to a tumortargeting mAb is a valid and versatile approach to generate novel immunotoxins by showing that (Q)-hRS7 (a) can be produced in a mammalian host and purified to homogeneity, (b) retains the binding specificity and affinity of hRS7, as well as the RNase activity of Rap, (c) suppresses the proliferation of various Trop-2-expressing cancer cell lines at nanomolar concentrations in vitro, and (d) inhibits the growth of a human lung tumor xenograft in vivo.…”

Milatuzumab–SN-38 Conjugates for the Treatment of CD74+ Cancers

“…Fab' internalization occurs just as rapidly as immunoglobulin G (IgG) binding, indicating that bivalent binding is not required (3,10). Later studies with a complementarity-determining region (CDR)-grafted version of murine LL1, milatuzumab (hLL1), found that the antibody could alter B-cell proliferation, migration, and adhesion molecule expression (8,11,12), but the exceptional internalization properties of the anti-CD74 antibody made it an efficient carrier for the intracellular delivery of cancer therapeutics (13)(14)(15)(16). On the basis of preclinical efficacy and toxicology results, phase I clinical trials with milatuzumab in multiple myeloma (17), as well as milatuzumab-doxorubicin in multiple myeloma, non-Hodgkin lymphoma, and chronic lymphocytic leukemia, have been initiated.…”