Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
2000-11-28
2004-05-11
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C536S023100, C536S023200, C435S320100, C424S093210
Reexamination Certificate
active
06733998
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a biosynthetic gene cluster of
Micromonospora echinospora
spp. calichensis. In particular, the calicheamicin biosynthetic gene cluster contains genes coding for proteins and enzymes used in the biosynthetic pathway and construction of calicheamicin's aryltetrasaccharide and aglycone, and the gene conferring calicheamicin resistance. The present invention also relates to isolated genes of the biosynthetic cluster and their corresponding proteins. In addition, the invention relates to DNA hybridizing with the calicheamicin gene cluster and the isolated genes of that cluster. The invention also relates to expression vectors containing the biosynthetic gene cluster, the individual genes, or functional variants thereof.
BACKGROUND OF THE INVENTION
The enediyne antibiotics, which were discovered in the 1980's, have long been appreciated for their novel molecular architecture, their remarkable biological activity, and their fascinating mode of action. Enediyne antibiotics were originally derived by fermentation of microorganisms, including Micromonospora, Actinomadura, and Streptomyces. Rothstein, D. M.,
Enediyne Antibiotics as Antitumor Agents
, p. 2 (1995). As a class, the enediyne antibiotics have been referred to as the most potent and highly active antitumor reagents yet discovered. Rothstein, D. M.,
Enediyne Antibiotics as Antitumor Agents
, preface (1995).
To date, at least twelve members of this family of antibiotics have been discovered, all of which fall roughly into two categories. The members of the first category of enediynes are classified as chromoprotein enediynes because they possess a novel 9-membered ring chromophore core structure, which also requires a specific associated protein for chromophore stabilization. The members of the second category of enediyne are classified as non-chromoprotein enediynes. These enediynes contain a 10-membered ring, which requires no additional stabilization factors. This enediyne ring structure is often referred to as the “warhead.” The warhead induces DNA damage, which is frequently a double-stranded cleavage and appears to be irreparable. This type of DNA damage is usually nonrepairable for the cell and is most often lethal. Because of these remarkable chemical and biological properties, there has been an intense effort by both the pharmaceutical industry and academia to study these substances with the goal of developing new and clinically useful therapeutic anti-tumor agents.
The 9-membered ring chromoprotein enediyne subfamily is comprised of: neocarzinostatin from
Streptomyces carzinostaticus
, (Myers, A. G., et al.,
J. Am. Chem. Soc
., 110, 7212-7214 (1988)); kedarcidin from Actinomycete L585-6, (Leet, J. E., et al.,
J. Am. Chem. Soc
., 114, 7946-7948 (1992)), N1999A2 from
Streptomyces globisporus
, (Yoshida, K., et al.
Tetrahedron Lett
., 34, 2637-2640 (1993)), maduropeptin from
Actinomadura madurea
, (Schroeder, D. R., et al.,
J. Am. Chem. Soc
., 116, 9351-9352 (1994)); N1999A2 from Streptomyces sp. AJ9493, (Schroeder, D. R., et al.,
J. Am. Chem. Soc
., 116, 9351-9352 (1994)); actinoxanthin from Actinomyces globisporus, (Khokhlov, A. S., et al.,
J. Antibiot., XXII
, 541-544 (1969)); largomycin from
Streptomyces pluricolorescens
, (Yamaguchi, T., et al.,
J. Antibiot., XXIII
, 369-372 (1970)); auromomycin from
Streptomyces macromomyceticus
, (Yamashita, T., et al.,
J. Antibiot., XXXII
, 330-339 (1979)), and sporamycin from
Streptosporangium pseudovulgare
, (Komiyama, K, et al.,
J. Antibiot
., XXX, 202-208 (1977)), all of which are believed to possess a novel bicylo[7.3.0.]dodecadiynene chromophore core structure essential for biological activity. In addition, with the exception of N1999A2, a required apoprotein acts as a stabilizer and specific carrier for the unstable chromophore, and for its transport and interaction with target DNA.
The non-chromophore enediyne subfamily is comprised of calicheamicin from
Micromonospora echinospora
spp. calichensis; namenamicin from
Polysyncraton lithostrotum
; esperamicin from
Actinomadura verrucosospora
; and dynemicin from
Micromonospora chersina.
Enediyne antibiotics have potential as anticancer agents because of their ability to cleave DNA; however, many of these compounds are too toxic to be used currently in clinical studies. Today, only calicheamicin is known to be currently used in clinical trials; and it has provided promising results as an anticancer agent. For example, MyloTarg™, a calicheamicin-antibody conjugate also known as CMA-676 was approved by the FDA in January of 2000 to treat acute myelogenous leukemia. The enediynes also potentially have utility as anti-infective agents, provided that toxicity can be managed.
Calicheamicin has two distinct structural regions: the aryltetrasaccharide and the aglycone (also known as the warhead). The aryltetrasaccharide displays a highly unusual series of glycosidic, thioester, and hydroxylamine linkages and serves to deliver the drug primarily to specific tracts (5′-TCCT-3′ and 5′-TTTT-3′) within the minor groove of DNA when those sequences are available. However, specificity is also context-dependent. The aglycone of calicheamicin consists of a highly functionalized bicyclo[7.3.1]tridecadiynene core structure with an allylic trisulfide serving as the triggering mechanism. McGahren, W. J.,et al.,
Enediyne Antibiotics as Antitumor Agents
, pp. 75-86 (1995). Once the aryltetrasaccharide is firmly docked, aromatization of the bicyclo[7.3.1]tridecadiynene core structure, via a 1,4-dehydrobenzene-diradical, results in the site specific oxidative double strand scission of the targeted DNA. Zein, N., et al.,
Science
, 240, 1198-1201 (1988). The aglycone undergoes a reaction that yields carbon-centered diradicals, which are responsible for DNA cleavage.
This activity of calicheamicin has sparked considerable interest in the pharmaceutical industry culminating in the recent FDA approval of the calicheamicin-antibody conjugate MyloTarg™ (CMA-676) to treat acute myelogenous leukemia (AML). Additionally, similar strategies have been used in phase I trials to treat breast cancer. A massive program to examine calicheamicin conjugated to alternative delivery systems has also recently been undertaken. Hamann, P. R., et al., 87th
Annual Meeting of the American Association of Cancer Research
, Washington, D.C., pp. 471 (1996); Hinman, L. M., et al.,
Cancer Res
., 53, 3336 (1993); Hinman, L. M., et al.,
Enediyne Antibiotics as Antitumor Agents
, pp. 87-105 (1995); Sievers, E. L., et al.,
Blood
, 93, 3678-3684 (1999); Siegel, M. M., et al.,
Anal. Chem
., 69, 2716-2726 (1997); Ellestad, G. personal communication.
The biological activity and molecular architecture of calicheamicin has also prompted a search for potentially useful analogs. Of the numerous laboratories producing synthetic analogs, one group has produced a novel calicheamicin &ggr;
1
1
shown to effectively suppress growth and dissemination of liver metastases in a syngeneic model of murine neuroblastoma. Lode, H. N., et al.,
Cancer Res
., 58, 2925-2928 (1998); Wrasidlo, W., et al.,
Acta Oncologica
, 34, 157-164 (1995). In addition to synthesizing calicheamicin analogs, random mutagenesis of
M. echinospora
and screening for mutant strains with improved biosynthetic potential has also been pursued. Rothstein, D. M.,
Enediyne Antibiotics as Antitumor Agents
, pp. 107-126 (1995).
The first total synthesis of calicheamicin was reported by Nicolaou and coworkers in 1992. Synthesizing this complex antibiotic, though, presents many disadvantages. For example, Nacelle's procedure only provides approximately a 0.007% yield and requires 47 steps. Halcomb, R. L., Enediyne Antibiotics as Antitumor Agents, pp. 383-439 (1995). Thus, the total synthesis of calicheamicin remains secondary to the isolation of calicheamicin from large fermentations of
M. echinospora
. Therefore, methods to produce mass amounts of calicheamicin and potentially useful
Kenyon & Kenyon
Ketter James
Sloan-Kettering Institute for Cancer Research
Sullivan Daniel M.
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