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
1997-10-20
2001-03-20
Fitzgerald, David L. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023520, C530S351000, C424S085400, C424S085700
Reexamination Certificate
active
06204022
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of reducing the toxicity of human interferon-alpha, to low-toxicity human interferon-alpha analogs, and to the therapeutic uses of these analogs.
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BACKGROUND OF THE INVENTION
The interferons (IFNs) have been classified into two distinct groups: type I interferons, including IFN&agr;, IFN&bgr;, IFN&tgr;, and IFN&ohgr; (also known as IFN&agr;II); and type II interferons, represented by IFN&ggr; (reviewed by DeMaeyer, et al., 1988). In humans, it is estimated that there are at least 17 IFN&agr; non-allelic genes, at least 2 IFN&bgr; non-allelic genes, and a single IFN&ggr; gene.
IFN&agr;'s have been shown to inhibit various types of cellular proliferation. IFN&agr;'s are especially useful against hematologic malignancies such as hairy-cell leukemia (Quesada, et al., 1984). Further, these proteins have also shown activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors and ovarian cancers (Bonnem, et al., 1984; Oldham, 1985). The role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has also been investigated (Benoit, et al., 1993).
IFN&agr;'s are also useful against various types of viral infections (Finter, et al., 1991). Alpha interferons have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, et al., 1991; Kashima, et al., 1988; Dusheiko, et al., 1986; Davis, et al., 1989).
Significantly, however, the usefulness of IFN&agr;'s has been limited by their toxicity: use of interferons in the treatment of cancer and viral disease results in serious side effects, such as fever, chills, anorexia, weight loss, and fatigue (Pontzer, et al., 1991; Oldham, 1985). These side effects often require (i) the interferon dosage to be reduced to levels that limit the effectiveness of treatment, or (ii) the removal of the patient from treatment. Such toxicity has reduced the usefulness of these potent antiviral and antiproliferative proteins in the treatment of debilitating human and animal diseases.
SUMMARY OF THE INVENTION
In one aspect, the invention includes methods for reducing the toxicity of human IFN&agr; (HuIFN&agr;). The method comprises substituting one or more of the amino acids at positions 19, 20, 22, 24, and 27 of mature HuIFN&agr; with an amino acid effective to substantially reduce the specific toxicity of the polypeptide when exposed to human mononuclear cells in culture. The majority of the amino acid residues 1-27 in mature HuIFN&agr; remains unchanged.
In one embodiment, the method includes substituting nonconserved amino acids for one or more of the amino acids at positions 19, 20, 22, and 27. In various embodiments, the substituting may include, but is not limited to: substituting a class III amino acid, in particular Asp, for the amino acid at position 19; substituting a class IV amino acid, in particular Arg, for the amino acid at position 20; substituting a class III amino acid, in particular Asn, for the amino acid at position 22; and substituting a class IV amino acid, in particular His, for the amino acid at position 27. In another embodiment, the substituting may include substituting a class V amino acid, in particular Leu, for the amino acid at position 24.
In another embodiment, the method comprises substituting the sequence of mature HuIFN&agr; between residues 19-27, with a 9-mer defined by SEQ ID NO:2. In particular, the sequence of mature HuIFN&agr; between residues 19-27 is SEQ ID NO:1. The 9-mer SEQ ID NO:2 corresponds to residues 19-27 of mature ovine interferon-tau (OvIFN&tgr;) and contains residues non-identical to mature HuIFN&agr; at positions 19, 20, 22, 24, and 27. In another embodiment, the method comprises substituting the sequence of HuIFN&agr; between residues 11-27 with a 17-mer defined by SEQ ID NO:4. In particular, the sequence of mature HuIFN&agr; between residues 11-27 is SEQ ID NO:3. The 17-mer SEQ ID NO:4 corresponds to residues 11-27 of mature OvIFN&tgr;, and contains residues non-identical to HuIFN&agr; at positions 11, 13, 14, 16, 19, 20, 22, 24, and 27. In another embodiment, the method comprises substituting the sequence of HuIFN&agr; between residues 6-27 with a 22-mer defined by SEQ ID NO:6. In particular, the sequence of mature HuIFN&agr; between residues 6-27 is SEQ ID NO:5. The 22-mer SEQ ID NO:6 corresponds to residues 6-27 of mature OvIFN&tgr;, and contains residues non-identical to HuIFN&agr; at positions 6, 7, 8, 11, 13, 14, 16, 19, 20, 22, 24, and 27.
In a related aspect, the invention includes a method for reducing the toxicity of HuIFN&agr;. The method includes substituting, for one or more of the amino acids at positions 19, 20, 22, 24, and 27 of mature HuIFN&agr;, an amino acid effective to substantially reduce the specific toxicity of the polypeptide in mononuclear cells in culture, where the mature HuIFN&agr; sequence between residues 28-166 is substantially unchanged. In one embodiment, said substituting is accomplished by substituting the sequence of HuIFN&agr; between residues 1-27 with the 27-mer defined by SEQ ID NO:8. In particular, the sequence of mature HuIFN&agr; between residues 1-27 is SEQ ID NO:7. The 27-mer SEQ ID NO:8 corresponds to residues 1-27 of mature OvIFN&tgr;, and contains residues non-identical to mature HuIFN&agr; at positions 2, 4, 5, 6, 7, 8, 11, 13, 14, 16, 19, 20, 22, 24, and 27.
In another aspect, the invention includes a low-toxicity human IFN&agr; analog for use in human therapy. This analog comprises a mature HuIFN&agr; protein having, at one or more of the amino acid positions 19, 20, 22, 24, and 27, a substituted amino acid, and the majority of the amino acid residues 1-27 in the analog are native HuIFN&agr; residues. The analog is characterized as having a substantially reduced specific toxicity relative to native human IFN&agr;, as evidenced by an increased viability of mononuclear cells in culture.
In one embodiment, the analog contains a nonconserved amino acid substitution at one or more of the positions 19, 20, 22, and 27. In various embodiments, the substituted amino acid may include, but is not limited to: a class III amino acid, in particular Asp, for the amino acid at position 19; a class IV amino acid, in particular Arg, for the amino acid at position 20; a class III amino acid, in particular Asn, for the amino acid at position 22; and a class IV amino acid, in particular His,
Campos Jackeline
Johnson Howard M.
Pontzer Carol H.
Subramaniam Prem S.
Villarete Lorelie H.
Fitzgerald David L.
Iota Pi Law Group
Mohr Judy M.
Pepgen Corporation and University of Florida
Petithory Joanne
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