Method of producing mouse and human endostatin

Details Method of producing mouse and human endostatin Method of producing mouse and human endostatin

1999-01-14

2003-11-25

Low, Christopher S. F. (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252330, C435S252500, C435S254200, C435S348000, C435S325000, C435S366000, C435S320100, C536S023100, C530S350000

Reexamination Certificate

active

06653098

ABSTRACT:

FIELD OF THE INVENTION
Methods for producing mouse and human endostatin are disclosed. Methods for refolding and purifying endostatin from inclusion bodies expressed in bacteria and nucleic acids encoding full-length and truncated forms of endostatin are also disclosed.
BACKGROUND OF THE INVENTION
Angiogenesis
Angiogenesis, the growth of new blood vessels, plays an important role in cancer growth and metastasis. In humans, the extent of vasculature in a tumor has been shown to correlate with the patient prognosis for a variety of cancers (Folkman, J.,
Seminars in Medicine of the Beth Israel Hospital
, Boston 333(26): 1757-1763, 1995; Gasparini, G.,
European Journal of Cancer
32A(14): 2485-2493, 1996; Pluda, J. M.,
Seminars in Oncology
24(2): 203-218, 1997; Norrby, K, APMIS 105: 417-437, 1997). In normal adults, angiogenesis is limited to well controlled situations, such as wound healing and the female reproductive system (Battegay, E. J.,
J Mol Med
73:-333-346, 1995; Dvorak, H. F,
New Engl J Med
, 315: 1650-1659, 1986).
Animal studies suggest that tumors can exist in a dormant state, in which tumor growth is limited by a balance between high rates of proliferation and high rates of apoptosis (Holmgren, L. et al.,
Nat. Med
. (N. Y.) 1(2): 149-153, 1995; Hanahan, D. et al.,
Cell
86(3): 353-364, 1996). The switch to an angiogenic phenotype allows tumor cells to escape from dormancy and to grow rapidly, presumably as the result of a decrease in the apoptotic rate of the tumor cells (Bouck, Cancer Cells, 2(6): 179-185, 1990; Dameron et al,
Cold Spring Harb Symp Quant Biol
, 59: 483-489, 1994). The control of angiogenesis is thought to be a balance between factors which promote new vessel formation and anti-angiogenic factors with suppress the formation of a neovasculature (Bouck, N. et al.,
Advances in Cancer Research
69: 135-173, 1996; O'Reilly et al., Cell 79(2): 315-328, 1994).
A variety of pro-angiogenic factors have been characterized including basic and acid fibroblast growth factors (bFGF and aFGF) and vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) (Potgens, A. J. G. et al.,
Biol. Chem. Hoppe-Seyler
376: 57-70, 1995; Ferrara, N.,
European Journal of Cancer
32A(14): 2413-2442, 1996; Bikfalvi, A. et al.,
Endocrine Reviews
18: 26-45, 1997). Several endogenous anti-angiogenic factors have also been characterized, including angiostatin (O'Reilly et al.,
Cell
79(2): 315-328, 1994), endostatin (O'Reilly et al,
Cell
88(2): 277-285, 1997), interferon-alpha. (Ezekowitz et al,
N. Engl. J. Med
., May 28, 326(22) 1456-1463, 1992), thrombospondin (Good et al,
Proc Natl Acad Sci
USA 87(17): 6624-6628, 1990; Tolsma et al., J Cell Biol 122(2): 497-511, 1993), and platelet factor 4 (PF4) (Maione et al, Science 247(4938): 77-79, 1990).
Many angiogenic inhibitors are in clinical development (See Shawver et al.,
Drug Discovery Today
2(2): 50-63, 1997, and references therein). Polypeptides such as interferon alpha and platelet factor 4 are in clinical trials. Angiostatin, soluble Flt-1 receptor, and bactericidal/permeability increasing protein derivative 23 are in preclinical studies. Monoclonal antibodies such as humanized anti-a
v
b
3
antibody (LM609), anti-VEGF, and anti-Flk-1 monoclonal antibody (DC101) are also in preclinical studies. Tecogalan (DS4152), a sulfated polysaccharide-peptidoglycan complex is in clinical trials, and bFGF carbohydrate inhibitor (GM1474) and glyceptor mimetic inhibitor of bFGF (GL14.2) are in preclinical studies. The antibiotic AGM1470 (TNP470), a fumagillin analog, and Suramin, a polyanionic compound are in clinical trials. Small molecule inhibitors such as urokinase receptor antagonists, inhibitors of phospatidic acid, inhibitors of Flk-1, and inhibitors of VEGF-F11 binding are all in preclinical studies. Thalidomide, and its analogues, and matrix metalloproteinase inhibitors, such as Batimastat/Marimastat, are in clinical trials. Oligonucleotides, such as ribozymes that target VEGF receptors and VEGF anti-sense oligonucleotides, are also in preclinical trials.
Anti-angiogenic therapy may offer several advantages over conventional chemotherapy for the treatment of cancer. Anti-angiogenic agents have low toxicity in preclinical trials and development of drug resistance has not been observed (Folkman, J.,
Seminars in Medicine of the Beth Israel Hospital
, Boston 333(26): 1757-1763, 1995). As angiogenesis is a complex process, made up of many steps including invasion, proliferation and migration of endothelial cells, it can be anticipated that combination therapies may be most effective. In fact, combinations of chemotherapy with anti-angiogenic therapy have already shown promising results in pre-clinical models (Teicher, B. A. et al.,
Breast Cancer Research and Treatment
36: 227-236, 1995; Teicher, B. A. et al.,
European Journal of Cancer
32A(14): 2461-2466, 1996).
Endostatin
Endostatin is a 20 kDa protein derived from the C-terminal fragment of alpha 1 type collagen XVIII. Conditioned cell culture media from a hemangioendothelioma cell line (EOMA) was shown to contain a factor which inhibited endothelial cell proliferation in vitro (O'Reilly et al.,
Cell
88: 277-285, 1997). The factor responsible for this inhibition was named endostatin. A recombinant form of this protein expressed in baculovirus-infected insect cells inhibited the growth of metastases in the Lewis lung tumor model and an insoluble
E. coli
derived form of this protein was shown to be efficacious in preventing primary tumor growth in several tumor models (O'Reilly et al.,
Cell
88: 277-285, 1997; Boehm et al., Nature 390: 404-410, 1997).
Purification and Refolding of Endostatin
Although many types of expression systems have been developed over the past twenty years, bacterial systems, particularly those based on
E. coli
, are widely used for the production of proteins on an industrial scale. Vectors which permit high level expression and the ability to carry out fermentations at high cell densities and low cost, have contributed to the extensive development and use of
E. coli
-based expression systems. One significant problem, however, is the tendency of
E. coli
to form inclusion bodies which contain the desired recombinant protein. Inclusion body formation necessitates additional downstream processing, such as in vitro refolding, before biologically active proteins can be recovered. The tendency to form insoluble aggregates does not appear to correlate with factors such as size, hydrophobicity, subunit structure, or the use of fusion domains (Kane J. F. and Harley, D. L.,
Tibtech
6: 95, 1988). Inclusion body formation appears to be determined by the rates of protein synthesis, folding, aggregation, and proteolytic degradation, the solubility and thermodynamics of folding intermediates and native proteins, and the interactions of these species with chaperone proteins (Rainer Rudolph, In Protein Engineering: Principles and Practice, Edited by Jeffrey L. Cleland and Charles S. Craik, p 283-298, Wiley-Liss, Inc., New York, N.Y., 1996).
Inclusion bodies generally form in the cytoplasm of cells expressing a recombinant protein at high levels. They refract light when observed by phase contrast microscopy and thus are sometimes referred to as refractile bodies. The inclusion bodies are characterized by a relatively high specific density and can be pelleted from lysed cells by centrifugation. The formation of inclusion bodies may protect recombinant proteins from proteolysis as they do not easily disintegrate under physiological solvent conditions. High concentrations of denaturants, such as 6 M guanidine hydrochloride or 6-8 M urea, have been commonly used to solubilize the proteins present in inclusion bodies. A variety of inclusion body solubilization protocols have been compared (Fisher, B., Summer, L. and Goodenough, P.
Biotechnol. Bioeng
. 1: 3-13, 1992).
Although the desired foreign gene product is the main component of inclusion bodies, other host cell proteins such as small heat shock proteins, out

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