Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-08-05
2003-12-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S254220, C514S002600, C530S300000, C530S350000
Reexamination Certificate
active
06670148
ABSTRACT:
FIELD OF THE INVENTION
In one aspect, the present invention relates to methods for preparing bioactive polypeptides in an inactive form. In another aspect, the present invention relates to bioactive polypeptides such as neurotoxins, and to methods for the preparation of such neurotoxins. In yet another aspect, the invention relates to the use of inactivated neurotoxin compositions for the study and treatment of viral and neurological diseases.
BACKGROUND OF THE INVENTION
Bioactive polypeptides are typically obtained by either the recovery and purification of natural products, or by synthesis using its genetic counterpart. Typically, the polypeptides, whether purified from natural sources or synthesized using recombinant technology, are ultimately provided in a form having the intended bioactivity.
Occasionally, however, it is desirable to prepare otherwise bioactive polypeptides in their inactive form, in which they can be used for other in vivo purposes, such as the preparation of vaccines. In other situations, the bioactivity of the polypeptide itself may be a particularly toxic one, so as to make the recovery of the active polypeptide either unnecessary, or unduly difficult and dangerous.
Typically, even toxic polypeptides are first recovered in their native, active forms, and thereafter subjected to processes intended to either temper the bioactivity, or render the polypeptide completely inactive. Examples of such processes include heating the polypeptide (e.g., denaturation), oxidation (e.g., by peroxide, catalase treatment), and the like. Such processes, however, are typically non-specific in nature, generally irreversible, and potentially quite damaging to the polypeptide.
Certain proteins can be inactivated by the cleavage of disulfide linkages, for instance using a suitable reducing agent (e.g., 2-mercaptoethanol) to provide a corresponding pair of cysteine residues. Cleavage of disulfide linkages within a protein will typically result in the unfolding of the protein. Unless maintained in the cleaved and unfolded state (e.g., in the presence of urea), the disulfide bonds are often able to spontaneously reform, although not always pairing the same original residues (resulting in a malfolded product).
Ribonuclease, for instance, contains four disulfide bonds that can each be cleaved in the manner described above. Under appropriate conditions, the molecule can spontaneously reform in a manner that provides 95-100% of the original activity. On the other hand, if the three disulfide bonds of insulin are cleaved under similar conditions, the molecule will spontaneously reform to provide only 5-10% of the original activity. Hence, the linear amino acid sequence of a protein is not necessarily the sole determinant of the protein's folding pattern and activity.
The recovery of neurotoxins is a prime example of the difficulties involved in handling and using bioactive molecules. See, generally, “Cloning, Characterization, and Expression of Animal Toxin (Genes for Vaccine Development”, L. A. Smith, J. Toxicol.-Toxin Reviews, 9(2), 243-283 (1990). The Smith article describes, for instance, the related properties of a number of toxins from animal origin (p. 247), and the slow progress made to date in developing such vaccines.
The venom obtained from snakes such as those of the genus Naja has been found to contain a number of different physiologically active, and potentially useful, polypeptides having enzymatic and/or toxic effects. A number of these toxins have been purified and modified for the purpose of determining their molecular structure and mode of action.
In order to safely use such toxins, for instance, U.S. Pat. Nos. 3,888,977, 4,162,303 and 4,126,676 (each naming Sanders) disclose detoxified venom compositions. The compositions are detoxified by oxidation using catalase or peroxide, in a manner said to retain the neurotropic activity of the modified venom compositions. The Sanders patents discuss compositions derived from the venom of the Bungarus genus, the Naja genus and a combination of both genuses. Included in such patents are methods for determining the potency and a toxicity of such modified neurotoxins.
Neurotoxin polypeptides in their detoxified but neurotropically active form have been considered for the treatment of certain viral infections. Detoxified polypeptides have been considered, for instance, for use in the treatment of certain disorders such as the neurological disorder amyotrophic lateral sclerosis (“ALS”), a disease characterized by slow progressive degeneration of lower motor neurons. See, for instance, “The Use of Sanders Neurotoxoid I (Modified Snake Venom) in the Treatment of Recurrent Herpes Simplex of the Cornea: Progress Report”, Clark, W. B., et al.,
Southern Medical Journal
55(9):947-951 (1962).
A variety of polypeptides, including toxins, have also been cloned and expressed by genetic engineering. See, for instance, the above-cited Smith article, which (beginning at page 257) describes a number of efforts directed at cloning snake venom toxin genes.
Conventional methods for preparing inactive polypeptides (e.g., detoxified neurotoxins) continue to suffer from a number of drawbacks. Among these drawbacks are the contaminants that frequently accompany the detoxified preparations. Another drawback relates to the fact that neurotoxins, unlike most polypeptides of a similar size, tend to be quite soluble in solvents commonly used for protein precipitation, thereby limiting the usefulness of conventional purification techniques. Yet another drawback relates to the use of any nonspecific means for rendering a polypeptide biologically inactive, since such means can often lead to the destruction of all properties, of the molecule, including such desirable properties as immunogenicity and antiviral activity.
What is clearly needed is a method for the preparation of inactivated bioactive polypeptides, such as neurotoxins, in a manner that avoids the drawbacks associated with prior methods. To applicants knowledge, there have been no teachings in the art of the use of genetic engineering techniques, particularly in the manner provided herein, to prepare inactive forms of bioactive molecules such as neurotoxins.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method of preparing a parenteral composition comprising an inactivated bioactive polypeptide, the method comprising the steps of:
a) identifying a polypeptide having a biological activity dependent on the presence of one or more disulfide bridges in its tertiary structure,
b) preparing a cDNA strand encoding the polypeptide,
c) expressing the cDNA under conditions in which the polypeptide is recovered in an inactive form due to the failure to form one or more disulfide bridges, and
d) recovering the inactive polypeptide and formulating it into a composition suitable for parenteral administration to a host.
In a further aspect, the invention provides a method of administering a composition comprising an inactivated bioactive polypeptide to a host, comprising the step of providing the polypeptide in an inactive form and in a composition that facilitates its administration to the host. In a related aspect, the invention provides a host having administered such a composition.
In another aspect, the invention provides a composition comprising a bioactive polypeptide that has been rendered inactive by virtue of the failure to form one or more of its disulfide bridges. In a related aspect, the invention provides a composition for in vivo administration comprising a bioactive polypeptide that has been inactivated in the manner described herein.
The method can be used to prepare a variety of bioactive polypeptides, including “Group I neurotoxins” (namely, toxins affecting the presynaptic neurojunction), Group II neurotoxins (namely those affecting the postsynaptic neurojunction), and Group III neurotoxins (those affecting ion channels). cDNA sequences for such polypeptides are generally known, or can be determined using conventional techniques.
The cDNA can be expressed u
Mundschenk David D.
Smith Leonard A.
Achutamurthy Ponnathapu
BioTherapeutics, Inc.
Fredrikson & Byron , P.A.
Moore William W.
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