Potent and specific antitumor effects of an anti-CD22–targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma

Newton, Dianne L.; Hansen, Hans J.; Mikulski, Stanislaw M.; Goldenberg, David M.; Rybak, Susanna M.

doi:10.1182/blood.v97.2.528

Cited by 98 publications

(57 citation statements)

References 38 publications

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“…Using yeast tRNA as substrate, we estimated the k cat /K m (10 9 M −1 s −1 ) of rRap and (Q)-hRS7 to be 4.10 (±0.42) and 1.98, respectively. Thus, the catalytic efficiency of (Q)-hRS7 based on the concentration of Rap is ∼50% of rRap, which was similar to the reported 40% catalytic efficiency of LL2-onconase compared with the native Rap (8). A plot of the initial rates versus the concentrations of tRNA from a representative set of experiments is shown in Fig.…”

Section: Rnase Activitysupporting

confidence: 82%

“…RNase activity was also determined by measuring the amount of perchloric acid-soluble nucleotides formed using yeast tRNA (Invitrogen) as substrate (8). Each sample was prepared with RNase-free water (Ambion) in a 1.5-mL RNase-free Eppendorf tube to contain, in a final volume of 100 μL, 5 nmol/L (Q)-hRS7 or rRap, 10 mmol/L HEPES (pH 6.0), 200 μg/mL human serum albumin, and a predetermined concentration of tRNA ranging from 100 μg/mL (3.09 μmol/L) to 600 μg/mL (18.54 μmol/L).…”

Section: Yeast Trna Degradation Assaymentioning

confidence: 99%

“…Conjugation or fusion of Rap to a tumor-targeting antibody or antibody fragment is a promising approach to enhance its potency, as first demonstrated for LL2-onconase (8), a chemical conjugate comprising Rap and LL2, a murine anti-CD22 monoclonal antibody (mAb), and subsequently for 2L-Rap-hLL1-γ4P (9), a fusion protein comprising Rap and hLL1, a humanized anti-CD74 mAb (10). The method for generating 2L-Rap-hLL1-γ4P allowed us to develop a series of structurally similar immunotoxins, referred to in general as 2L-Rap-X, all of which consist of two Rap molecules, each connected through a flexible linker to the NH 2 terminus of one light chain (L chain) of an antibody of interest (X).…”

Section: Introductionmentioning

confidence: 99%

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

Chang

Gupta

Michel

et al. 2010

Molecular Cancer Therapeutics

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Ranpirnase (Rap), an amphibian RNase, has been extensively studied both preclinically and clinically as an antitumor agent. Rap can be administered repeatedly to patients without any untoward immune response, with reversible renal toxicity reported to be dose limiting. To enhance its potency and targeted tumor therapy, we describe the generation of a novel IgG-based immunotoxin, designated 2L-Rap(Q)-hRS7, comprising Rap (Q), a mutant Rap with the putative N-glycosylation site removed, and hRS7, an internalizing, humanized antibody against Trop-2, a cell surface glycoprotein overexpressed in variety of epithelial cancers. The immunotoxin was generated recombinantly by fusing Rap(Q) to each of the two hRS7 light (L) chains at the NH 2 terminus, produced in stably transfected myeloma cells, purified by Protein A, and evaluated by a panel of in vitro studies. The results, including size-exclusion high-performance liquid chromatography, SDS-PAGE, flow cytometry, RNase activity, internalization, cell viability, and colony formation, showed its purity, molecular integrity, comparable affinity to hRS7 for binding to several Trop-2-expressing cell lines of different cancer types, and potency to inhibit growth of these cell lines at nanomolar concentrations. In addition, 2L-Rap (Q)-hRS7 suppressed tumor growth in a prophylactic model of nude mice bearing Calu-3 human non-small cell lung cancer xenografts, with an increase in the median survival time from 55 to 96 days (P < 0.01). These results warrant further development of 2L-Rap(Q)-hRS7 as a potential therapeutic for various Trop-2-expressing cancers, such as cervical, breast, colon, pancreatic, ovarian, and prostate cancers. Mol Cancer Ther; 9(8); 2276-86. ©2010 AACR.

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Section: Rnase Activitysupporting

confidence: 82%

Section: Yeast Trna Degradation Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Chang

Gupta

Michel

et al. 2010

Molecular Cancer Therapeutics

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“…1). Ranpirnase is the smallest member of the ribonuclease (RNase A) superfamily, and appears to be a promising drug with broad clinical application in tumor treatment due to its moderate cytotoxicity, unique synergy, low immunogenicity and few side effects (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Between 1996 and 2004, Tamir Biotechnology, Inc. (formerly Alfacell Corporation) successively conducted clinical investigations regarding the effects of ranpirnase on breast cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer and malignant mesothelioma, for which the therapeutic effect was the most significant with few side effects.…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression, different downstream processing of the disulfide-rich anti-tumor peptide Ranpirnase and its effect on the growth of human glioma cell line SHG-44

Wang

Guo

2013

Biomedical Reports

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Abstract. Ranpirnase (Onconase) is a frogspawn-derived disulfide-rich peptide with ribonuclease activity that may be used for tumor treatment. In the present study, we established an efficient approach for preparing mature ranpirnase which may be used for research and therapeutic purposes. The designed ranpirnase precursors carried a 6xHis-tag and were recombinantly expressed in Escherichia coli. After S-sulfonation, the precursors were purified by immobilized metal-ion affinity chromatography. Following removal of the tag by aminopeptidase cleavage, cyclization and in vitro oxidative refolding, the mature ranpirnase was obtained with considerable yield, and the yield of mature ranpirnase was ~50-60 mg per liter cultures. In addition, ranpirnase inhibited the growth of human glioma cells SHG-44 in a dose-dependent manner. Thus the present study has provided an efficient approach for the preparation of active ranpirnase and its analogues for future studies.

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“…It is logical to predict that the ability of CD22 to modulate BCR-dependent signaling may be regulated by the spatial relationship between these receptors in the plasma membrane, or by regulating the level of CD22 expression on the cell surface, or both. Studies have shown that CD22 is an effective target for treatment of B cell tumors because binding of mAbs leads to efficient internalization and intracellular routing of immunotoxins (25)(26)(27). CD22 undergoes constitutive internalization and is subsequently degraded in an acidic intracellular compartment without recycling back to the surface (28).…”

mentioning

confidence: 99%

The B Cell Coreceptor CD22 Associates with AP50, a Clathrin-Coated Pit Adapter Protein, Via Tyrosine-Dependent Interaction

John

Herrin

Raman

et al. 2003

The Journal of Immunology

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The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr843 and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr843 or Tyr863 is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of α-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab′)2 blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.

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Potent and specific antitumor effects of an anti-CD22–targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma

Cited by 98 publications

References 38 publications

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

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

Recombinant expression, different downstream processing of the disulfide-rich anti-tumor peptide Ranpirnase and its effect on the growth of human glioma cell line SHG-44

The B Cell Coreceptor CD22 Associates with AP50, a Clathrin-Coated Pit Adapter Protein, Via Tyrosine-Dependent Interaction

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