Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Utility Patent
1998-07-13
2001-01-02
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S071300, C435S471000, C435S252300, C435S348000, C435S320100, C530S412000, C530S416000
Utility Patent
active
06168932
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions and methods for large scale production of a fusion toxin. In particular, the invention relates to the large scale production of diphtheria toxin fused to a moiety selected to target the toxin to a specific cell or tissue, such as a tumor cell.
BACKGROUND OF THE INVENTION
Acute myeloid leukemia (AML) is the most common form of leukemia found in adults and the second most common leukemia affecting children. Despite intensive multimodality treatment programs, including chemotherapy and bone marrow transplantation, more than one-half of patients with AML die because of therapy-refractory or recurrent leukemia. Consequently, the development of effective new agents capable of killing multidrug resistant leukemia cells has emerged as an exceptional focal point for translational research in treatment of AML.
Recombinant DNA technology is now used to generate fusion toxins, providing a means for producing these therapeutic biomolecules with consistent characteristics. For example, recombinant DNA techniques have been used to produce diphtheria toxin (DT) fused to targeting moieties including interleukin (IL)-2, IL-4, IL-6, epidermal growth factor (EGF), and the melanocyte-stimulating hormone (MSH) in order to target this toxin to specific tumor cells expressing the receptor for the targeting moiety. Clinical trials using DT
ct
IL-2 fusion toxins have provided encouraging evidence for partial or complete remission of disease in patients with IL-2 receptor positive lymphoid malignancies.
DT
ct
GMCSF is a genetically engineered recombinant fusion toxin that directs the lethal diphtheria toxin (DT) to high affinity granulocyte-macrophage colony stimulating factor (GMCSF) receptors (R) present on specific tumor cells, including, among others, AML cells. DT
ct
GMCSF preserves the portions of DT that include the lethal catalytic ADP-ribosyltransferase domain (c domain) and the contiguous portion of DT that is associated with translocation across cellular membranes (t domain). The native receptor binding domain of DT is replaced with human GMCSF in the construction of the DT
ct
GMCSF fusion toxin. The preparation and efficacy of a fusion toxin such as DT
ct
GMCSF has been described, for example, in U.S. Pat. Nos. 5,744,580, 5,756,699, 5,677,274, and 5,681,810.
DT
ct
GMCSF is selectively cytotoxic to a wide range of AML cells, including those with multidrug resistance. DT
ct
GMCSF induces rapid apoptotic death in chemotherapy-resistant AML cell lines and in primary leukemia cells from therapy-refractory AML patients. At nontoxic dose levels, DT
ct
GMCSF is superior to standard chemotherapeutic agents such as ARA-C and adriamycin, resulting in 60% long-term survival of severe combined immunodeficient (SCID) mice challenged with an otherwise invariably fatal dose of xenografted human AML cells. Importantly, systemic exposure of cynomolgus monkeys to levels of DT
ct
GMCSF which were found to be therapeutic in the SCID mouse xenograft model of human AML, can be achieved without any significant nonhematological toxicity. Taken together, these preclinical studies indicate that DT
ct
GMCSF fusion toxin has clinical potential for more effective treatment of therapy-refractory AML patients.
DT
ct
GMCSF is selectively cytotoxic to GMCSF receptor (R) positive acute myeloid leukemia (AML) cells both in vitro and in vivo (see for example, Perentesis et al., 1997,
Clinical Cancer Research,
3:347-355; and Perentesis et al., 1997,
Clinical Cancer Research,
3:2217-2227), however, its clinical development has been hampered by the inability to produce large amounts of the cytotoxin in traditional host cell systems. Previous expression attempts have resulted in very low expression levels, requirements for solubilization with guanidine hydrochloride and subsequent refolding, and concerns about bacterial endotoxin contamination. In addition, DT
ct
GMCSF is toxic to most cellular hosts, causing the host cell death before significant amounts of toxin can be harvested. Since clinical trials and commercial production require relatively large amounts of DT
ct
GMCSF to be produced, a more efficient system for large scale production of DT
ct
GMCSF is needed.
SUMMARY OF THE INVENTION
It has now been discovered that large amounts of biologically active DT
ct
GMCSF can be produced using insect cell hosts. The fusion toxin produced in the insect cells is soluble, and can be easily purified to homogeneity by column chromatography. Surprisingly, DT
ct
GMCSF was expressed in the cytoplasm of insect cells at a very high level. Approximately 8-10 mg/L of purified DT
ct
GMCSF is routinely obtained from 1 liter of insect cell culture (about 10
9
cells). Production of DT
ct
GMCSF in insect cells thus provides a suitable means for generating sufficient amounts of DT
ct
GMCSF for Phase I/II clinical trials and is amenable to further scale-up for commercial production.
The fusion protein is preferably expressed in an insect cell system using the baculovirus expression vector system. The fusion toxin can be isolated from the insect cells in high yield using a solubilization buffer that preferably includes a surfactant, for example Tergitol NP-40, followed by ion exchange chromatography. Preferably, a first column is neutral or slightly basic, at about pH 7.5 (preferably 7.4) and a second column is acidic at about pH 4-5 (preferably 4.1). This process results in simpler purification of high yield of biologically active fusion protein.
The invention also includes baculovirus expression vectors containing a nucleic acid sequence encoding the fusion toxin and insect cells useful to express large quantities of fusion toxin. Preferred expression vectors include a baculovirus expression system containing a DNA sequence encoding a DT
ct
-targeting moiety fusion toxin. Insect cell cultures transformed or transfected with an expression vector provide for expression of fusion toxin, preferably in high yield.
REFERENCES:
patent: WO 96/38571 (1996-12-01), None
Arceci, R. J., “Clinical Significance of P-Glycoprotein in Multidrug Resistance Malignancies [editorial] [see comments]”,Blood,81, 2215-2222 (1993).
Au, L. C., et al., “Secretory Production of Bioactive Recombinant Human Granulocyte- Macrophage Colony Stimulating Factor by a Baculovirus Expression System”,J. Biotechnol,51, 107-113 (1996).
Bendel, A. E., et al., “A Recombinant Fusion Toxin Targeted to the Granulocyte-Macrophage Colony-Stimulating Factor Receptor”Leuk Lymphoma,25, 257-270 (1997).
Bradford, M. M., “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding”,Anal Biochem.,72, 248-254 (1976).
Brocks, B., “A TNF Receptor Antagonistic scFv, Which is Not Secreted in Mammalian Cells, is Expressed as a Soluble Mono- and Bivalent scFv Derivative in Insect Cells”,Immunotechnology.,3, 173-184 (1997).
Chan, H. S., et al., “Multidrug Resistance. Clinical Opportunities in Diagnosis and Circumvention”,Hematol Oncol Clin North Am.,8, 383-410 (1994).
Chiou, C. J., et al., “Expression of Human Granulocyte-Macrophage Colony-Stimulating Factor Gene in Insect Cells by a Baculovirus Vector”.FEBS Lett,259, 249-253 (1990).
Collins, S. J., “The HL-60 Promyelocytic Leukemia Cell Line: Proliferation, Differentiation, and Cellular Oncogene Expression”,Blood70, 1233-1244 (1987).
Espevik, T., et al., “A Highly Sensitive Cell Line, WEHI 164 Clone 13, for Measuring Cytotoxic Factor/Tumor Necrosis Factor from Human Monocytes”,J Immunol Methods,95, 99-105 (1986).
Frankel, A. E., “Characterization of a Ricin Fusion Toxin Targeted to the Interleukin-2 Receptor”,Protein Eng.,9, 913-919 (1996).
Frankel, A., et al., “IL2-Ricin Fusion Toxin is Selectively Cytotoxic In Vitro to IL2 Receptor-Bearing Tumor Cells”,Bioconjug Chem.,6, 666-672 (1995).
Fraser, A., et al., “A License to Kill”,Cell.,85, 781-784 (1996).
Green, L. M., et al., “Rapid Colorimetric Assay for Cell Viability: Application to the Quantitation of Cytotoxic and Growth Inhibitory Lymphokines
Rostovstev Alexander
Uckun Fatih M.
Williams Mark D.
Hamud Fozia
Merchant & Gould P.C.
Mertz Prema
Parker Hughes Institute
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