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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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  • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

• This invention relates to a microorganism that is a mutant bacterial strain of the species Actinosynnema pretiosum, designated strain PF4-4 (ATCC PTA-3921), being capable of producing maytansinoid ansamitocins such as ansamitocin P-3 in improved yield compared to previous known strains, and to methods of producing such maytansinoid ansamitocins from said strain PF4-4.
• Bacteria of the species Actinosynnema pretiosum produce cytotoxic maytansinoid antibiotics (Higashide et al. Nature 270, 721-722, 1977). Bacteria of this species were originally classified and deposited as Nocardia sp., however subsequent characterization demonstrating the absence of any mycolic acids, cell wall type ffl/C (maso-diaminopimelic acid and no diagnostically important carbohydrates), a lack of sporangia, and the formation of motile elements, indicated that these strains are members of the genus Actinosynnema (Hasegawa, et al. "Motile Actinomycetes: Actinosynnema pretosium subsp.
• the bacterially produced maytansinoids are called ansamitocins, and comprise a group of antiturnor benzenoid ansamycin antibiotics that are distinguished from one another by their substitutions at the C-3 and C-14 positions, as shown by substituents R and R ⁇ of formula (I).
• Several strains of Actinosynnema have been deposited, such as ATCC 315651, Actinosynnema pretiosum subsp. auranticum.
• the metabolic, physiological and maytansanoid-producing properties of ATCC 31565 are described in U.S. Patents 4,331,598 and 4,450,234 to Hasegawa et al., issued on May 25, 1982, and May 22, 1984, respectively.
• ATCC 31565 is a gram-positive bacterium that is capable of growth on a wide range of carbon sources and which produces principally a mixture of maytansinoids and of C-14- hydroxymethyl substituted maytansinoids that may be harvested from the growth medium in low yield.
• Maytansinoids were originally isolated from African plants (Kupchan et al. J. Amer. Chem. Soc. 94, 5294-5295, 1972). Production of maytansinoids from such sources was difficult because they were present in very small amounts.
• a maytansinoid-producing microorganism was subsequently isolated from sedge blades, which was classified as a new strain of the genus Nocardia, Nocardia sp. strain No.C-15003 (N-l). This strain was deposited as ATCC 31281, and is disclosed in U.S. Patent 4,137,230 to Hashimoto et al., issued Jan 30, 1979, and U.S. Patent 4,162,940 to Higashide et al., issued July 31, 1979. Purification of maytansinoids from this bacterium requires fewer steps and results in increased yield compared to purification from plant sources.
• a second maytansinoid-producing strain was isolated from sedge blades, named Nocardia sp. Strain No. C-14482 (N-1001), deposited as ATCC 31309. This strain is disclosed in U.S. Patent 4,292,309 to Higashide et al., issued September 29,1981.
• a third strain was derived from ATCC 31309, designated C-14482, by a process of mutagenesis. This third strain was named Nocardia sp. No. N-1231, and was deposited as ATCC 31565.
• Maytansinoids have potent cytotoxic activity and have demonstrated strong anti-tumor activity when delivered in conjugate form with a cell-binding agent.
• U.S. Patent 5,208,020 to Chari et al. discloses a cytotoxic agent comprising one or more maytansinoids linked to a cell targeting agent such as an antibody, whereby the maytansinoid is directed toward killing selected cell populations through the specific cell-binding agent.
• U.S. Patent 5,416,064 also to Chari at el., issued May 16, 1995, discloses new maytansinoids that are attached to cell-binding agents through cleavable disulfide linkages, whereby the maytansinoid is released intracellularly.
• These conjugates have pharmaceutical potential for the treatment of various cancers.
• the present invention provides a bacterial strain, deposited as ATCC PTA-3921, also termed herein "PF4-4," that produces increased amounts of maytansinoids.
• PF4-4 was obtained by mutation from parent strain N-1231 (ATCC 31565) using ultraviolet light (UN light), l-methyl-3-mtro-l-nitroso-guanidine (M ⁇ G), and selection for enhanced maytansinoid production.
• the invention comprises a mutated bacterial strain
• This embodiment of the invention is capable of producing more than 500 mg/L of ansamitocin P-3, which is a yield improvement of 5- to 10-fold compared to the parental
• This embodiment is further capable of producing substantial amounts of other ansamitocin species, for example ansamitocins P-2 and P-4. Furthermore, the relative amounts of specific ansamitocin species that are produced by this embodiment of the invention are capable of rational manipulation through the choice of carbon source used to support growth.
• This embodiment is capable of growth upon a wide variety of carbon sources, and, with the exception of its capability to produce increased amounts of maytansinoids, is substantially similar to the parental strain (ATCC 31565) with respect to its morphology, physical and metabolic characteristics.
• one object of the present invention is to provide a bacterial strain that is capable of enhanced maytansinoid production, whereby such maytansinoids are highly cytotoxic and can be used as therapeutic agents, for example in the form of a conjugate with a cell-specific component, in the treatment of many diseases, including cancer.
• a second object of the invention is to provide a bacterial strain that is capable of enhanced maytansinoid production such that maytansinoid may be produced in sufficient quantities to facilitate commercial development of said therapeutic agents.
• a third object is to provide a method for the production of maytansinoid ansamitocins from strain PF4-4 by culturing said strain in a growth medium comprising a suitable carbon source.
• the proportions of maytansinoid ansamitocins produced by this method may be predetermined by the choice of carbon source.
• FIG. 1 is a diagram showing the ansamitocin P-3 titer for 400 re-isolates from
• FIG. 2 is a diagram showing one method for the production of ansamitocin P-3 by shake flask fermentation of Actinosynnema pretiosum, mutant strain PF4-4.
• FIG. 3 is an HPLC chromatogram of a sample of broth extract from the fermentation of Actinosynnema pretiosum, mutant strain PF4-4. DETAILED DESCRIPTION OF THE INVENTION
• the present invention comprises a microorganism that is a mutant bacterial strain of the species Actinosynnema pretiosum, designated PF4-4 (ATCC PTA-3921), which is capable of producing maytansinoids, including ansamitocins such as ansamitocin P-3 and other ansamitocins in improved yields compared to previously known strains, including the parent strain (ATCC 31565).
• PF4-4 ATCC PTA-3921
• maytansinoids including ansamitocins such as ansamitocin P-3 and other ansamitocins in improved yields compared to previously known strains, including the parent strain (ATCC 31565).
• PF4-4 The bacterial strain, PF4-4, of the present invention was produced from parent strain ATCC 31565 by mutation using UN light, l-methyl-3-nitro-l-nitroso-guanidine (M ⁇ G) and selection, to derive a strain of genetically altered bacteria that produces substantially higher quantities of maytansinoids than the parental strain.
• M ⁇ G l-methyl-3-nitro-l-nitroso-guanidine
• PF4-4 is capable of producing more than 500 mg/L of ansamitocin P-3, which is 5-10 fold more than the amount produced by the parental strain under the same conditions.
• the Actinosynnema pretiosum strain of the present invention herein PF4-4, was deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville Maryland, on December 11, 2001, and has been accorded Accession No. ATCC PTA-3921.
• the parent strain ATCC 31565 from which the Actinosynnema pretiosum strain PF4-4 of the present invention is derived by mutation, is itself derived from Actinosynnema pretiosum strain ATCC 31309, as disclosed in U.S. Patent 4,292,309, to Higashide et al., issued September 29, 1981.
• bacteria of this species were originally classified and deposited as Nocardia sp., however subsequent characterization indicated that these strains are members of the genus Actinosynnema (Hasegawa, T. et al. "Motile Actinomycetes: Actinosynnema pretosium subsp.
• the term "maytansinoid” refers to the class of highly cytotoxic drugs first isolated from the east African shrub Maytenus ovatus, and further includes: maytansinol and naturally occurring C-3 esters of maytansinol (U.S. Patent 4,151,042); synthetic C-3 ester analogues of maytansinol (Kupchan et ah, J. Med. Chem. 21:31-37, 1978; Higashide at al., Nature 270:721-722, 1977; Kawai et al., Chem. Farm. Bull. 32:3441-3451; and U.S. Patent 5,416,064); C-3 esters of simple carboxylic acids (U.S.
• Patents 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598); and C-3 esters with derivatives of N-methyl-L-alanine U.S. Patents 4,137,230; 4,260,608; and Kawai et al, Chem. Pharm Bull. 12:3441, 1984).
• ansamitocin refers to various derivatives of ansamycin antibiotics (Hasegawa, T. et al. "Motile Actinomycetes: Actinosynnema pretosium subsp. pretosium sp nov., subsp. nov., and Actinosynnema pretosium subsp. auranticum susp. nov.” Int. J. System. Bacteriol. 33(2):314-320, 1983; Tanida et al. "Ansamitocin analogs from a mutant strain of nocardia. I. Isolation of the mutant, fermentation and antimicrobial properties.” J. Antibiotics 34: 489-495, 1981) represented by the following general formula (I):
• R represents, for example, hydrogen, acetyl, propionyl, isobutyryl, butyryl, isovaleryl, and the like, and wherein R ⁇ represents, for example, methyl, hydroxymethyl, and the like.
• ansamitocin P compounds with a methyl group at R t (C-14) in formula I Ansamitocin PHM compounds with a hydroxymethyl group at Ri (C-14) in formula I
• ansamitocin PDN compounds with a N-desmethyl ring structure and a methyl group at the Ri position of formula I In each class there are several members that are distinguished by different substituents R (C-3) in formula I.
• R (C-3) substituents
• ansamitocins are designated herein by the abbreviations given as follows:
• ansamitocin further encompasses isomers thereof, including isomers occurring at the C-3, C-4, C-9 and C-10 positions.
• strain PF4-4 of the present invention exhibits enhanced maytansinoid production.
• the morphological and metabolic characteristics of the parent strain are disclosed in U.S. Patent 4,450,234 to Hasegawa et al., issued May 22, 1984, and those portions describing said characteristics, including, but not limited to, columns 3-7 are hereby incorporated in their entireties by reference.
• the PF4-4 strain is obtained from the parent strain N-1231, ATCC 31565, by the following procedure.
• strain N-1231, ATCC 31565 exhibits an average production of ansamitocin P-3 of 60 mg/L, and no colony exhibits production of greater than 221 mg/L.
• Strain PF4-4 is preferably generated from ATCC 31565 in seven consecutive steps. These seven steps are: re-isolation; a first round of mutagenesis; re-isolation, preferably three times; UN mutagenesis, and M ⁇ G mutagenesis.
• UN mutagenesis is described in detail in EXAMPLE 1 (below).
• spores from an 8-day old slant culture of colony No. 15-55 are collected in water and macerated on a vortex mixture.
• the number of colony forming units (cfu) is determined and is typically found to be about 2 x IO 9 .
• Serially diluted samples containing varying numbers of cfu are then spread on agar plates and exposed to UN light from a germicidal lamp for different lengths of time. The killing rate for a 40 second exposure to such light is typically 99.9 %.
• Plates treated for varying lengths of time are incubated at 28 °C for 5-7 days and then colonies are selected and analyzed for ansamitocin P-3 production. The colony having the highest ansamitocin P-3 production is selected for use in the next step.
• MNNG mutagenesis is described in detail in EXAMPLE 2 (below).
• macerated spores are prepared as above, collected by centrifugation, and re-suspended in buffer containing lOO ⁇ g/mL MNNG.
• the mutagenesis reaction is stopped after about 30 min by the addition of excess sodium thiosulfate and the bacteria are then collected by centrifugation, washed, and plated on agar plates for determination of the survival rate and for further analysis.
• Table 5 shows the genealogy of strain PF4-4 and the production by intermediate isolates of ansamitocin P-3 (mg L) as assayed by HPLC (see EXAMPLE 6).
• the media used in Table 5 are given in Table 6A.
• the entry "d/n” represents the time of fermentation in days (d) and the number of cultures tested (n).
• PF4-4 Growth of the bacterial strain PF4-4 is performed under controlled conditions and can employ a wide variety of media and conditions.
• PF4-4 can be grown under similar conditions and with similar media to those described for ATCC 31565 or ATCC 31281 in issued U.S. Patents 4,137,230; 4,162,940 ' ; 4,331,598; 4,356,265; 4,450,234; and as described in Hatano et al., Agric. Biol. Chem. 48, 1721-1729, 1984.
• the strain PF4-4 tolerates a wide variety of carbon sources, which also support fermentative production of maytansanoids.
• Exemplary growth media are given in Tables 6A and 6B.
• Table 6A shows media that support growth of PF4-4 and which are utilized in Table 5.
• Table 6B shows further media suitable for the propagation and/or growth of PF4-4.
• Ansamitocin P-3 is the major product of PF4-4 and the parental strain ATCC 31565, under certain growth conditions. If the bacteria are grown in the presence of valine or isobutyric acid (see U.S. Patent 4,228,239) or isobutyl alcohol or isobutylaldehyde (see U.S. Patent 4,356,265) other ansamitocin compounds are present in minor amounts.
• ansamitocin P-3 is the predominant ansamitocin produced. Fermentation broths are diluted with ethanol or acetonitrile, vortexed, then centrifuged and the supernatant assayed for ansamitocin P-3 content.
• Ansatomycins are preferably fractionated and analyzed by reverse phase high performance liquid chromatography (hplc), but any suitable technique, such as, for example, MALDI-TOF or thin-layer chromatography may be used.
• HPLC high performance liquid chromatography
• fermentation broths are extracted with organic solvents, such as ethyl acetate, methylene chloride or chloroform, and the content of P-3 in the organic solvent is determined by reverse phase hplc as described in EXAMPLE 6.
• the macerated suspension typically contains 2 x IO 9 cfu.
• 0.1 mL of the suspension was plated on a CM4-1 agar plate (9.5 cm diameter), which was exposed to a suitable germicidal UN light source: the open agar plate was placed under a 15 W germicidal lamp at about 20 cm distance and exposed for 20-40 seconds to the UN light. (The killing rate for a 40 second exposure was about 99.9 %.)
• the exposed plates were cultivated at 28 °C for 5-7 days, and single colonies were then transferred to another CM4-1 agar plate and grown in grids of 16 colonies per plate. Colonies were then chosen for further evaluation.
• Spores from an 8-day old slant preparation were collected by washing the slant (slant size: 2.3 x 18 cm tube filled with 16-18 mL of CM4-1 agar) with 10 mL of water. Five mL of the water with the suspended spores were placed in a screw cap tube (size: 1.1 x 11 cm) containing 10 glass beads of 2.0 mm diameter for maceration. The tube was vortexed for five minutes and the spores were collected by centrifugation at 2100 x g for 15 min.
• the supernatant was discarded and the pellet was resuspended in 4 mL of sterile 0.05 M tris maleic acid buffer, pH 8.0 containing (w/v) 0.1 % ammonium sulfate, 0.01 % magnesium sulfate hepta-hydrate, 0.005 % calcium chloride dihydrate, 0.00025 % ferrous sulfate hepta- hydrate, and 100 ⁇ g/mL of MNNG.
• the suspension was vortexed for 30 min, then the reaction was stopped by the addition of 3 mL of a saturated sodium thiosulfate solution.
• the spores were collected by centrifugation, then resuspended in 5 mL of water. This suspension was used for selection and an appropriate dilution was smeared on a CM4-1 agar plate to determine the survival rate.
• a stored PF4-4 culture for example a lyophilized or frozen culture, was grown on
• CM4-1 agar plates at 28 °C for 5-7 days. Single colonies were then transferred to a second, gridded CM4-1 agar plate, (typically 16 colonies were transferred to a plate of 9.5 cm diameter), and the plate was incubated at 28 °C for 7 days, during which period colonies of 6- 15 mm in diameter grew. A single colony was then macerated by vortexing for 10 minutes in a closed tube containing ten 2-mm-diameter glass beads and 2 mL of water. Part of the colony suspension (0.5 mL) was then transferred to a 250 mL culture flask containing 30 L seed medium, VM4-1 (for composition, see Table 6b).
• the seed flask was incubated on a rotary shaker (220 rpm, 70 mm throw) at 28 °C for 48 hours, after which 1 mL of the seed growth suspension was transferred to a 250 mL culture flask containing 20 mL of fermentation medium FM4-4.
• the fermentation flask was incubated under the same conditions as the seed flask for 6 days, after which ansamitocin production was assayed as described in EXAMPLE 6 and was found to be 268 mg/L.
• a macerated colony suspension in 2 mL of water was prepared as described in EXAMPLE 3, then 0.2 mL of the suspension was inoculated onto a slant culture (slant size: 2.3 x 18 cm tube filled with 16-18 mL of CM4-1 agar) and incubated at 28 °C for 7 days.
• the slant was washed out with 10 mL of cryogen solution (10 % glycerol and 5 % lactose in water), which was then subjected to the maceration procedure described above.
• the macerated suspension was aliquoted (1.5 mL) into cryovials and frozen at 75 °C or in liquid nitrogen.
• Fermentation broth (0.25 mL) was transferred to a screw cap tube containing ethanol (4.75 mL). (Alternatively, 0.25 mL of whole fermentation broth was mixed with 2.25 mL of ethanol.) The solution was vortexed for ten minutes, then centrifuged at 2100 x g for ten minutes. The supernatant was removed and subjected to reverse phase hplc analysis. Preferably, a Symmetry Shield C8 column (3.6 x 150 mm) was used. The mobile phase was preferably water/acetonitrile/methanol at a ratio (v/v) of 55/35/10 and was preferably used at a flow rate of 1.0 mL/min. Chromatography was monitored by measuring UN absorption at 252 nm. A typical hplc trace of a broth extract is shown in Figure 3, in which ansamitocin P-3 elutes at about 12.2 minutes after the injection.

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