Title:  Low-toxicity human interferon-alpha analogs

United States Patent:   6,204,022

Assignee:  Pepgen Corporation and University of Florida ()

Filed:  October 20, 1997

The invention describes a method of reducing the cytotoxicity of interferon-alpha by making defined amino acid substitutions in the N-terminal portion of the polypeptide sequence. Also described are human interferon-alpha analogs with low cytotoxicity, and therapeutic applications of the low toxicity interferon-alpha analogs.

SUMMARY OF THE INVENTION

In one aspect, the invention includes methods for reducing the toxicity of human IFN.alpha. (HuIFN.alpha.). The method comprises substituting one or more of the amino acids at positions 19, 20, 22, 24, and 27 of mature HuIFN.alpha. 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.alpha. 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.alpha. between residues 19-27, with a 9-mer defined by SEQ ID NO:2. In particular, the sequence of mature HuIFN.alpha. 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.tau.) and contains residues non-identical to mature HuIFN.alpha. at positions 19, 20, 22, 24, and 27. In another embodiment, the method comprises substituting the sequence of HuIFN.alpha. between residues 11-27 with a 17-mer defined by SEQ ID NO:4. In particular, the sequence of mature HuIFN.alpha. between residues 11-27 is SEQ ID NO:3. The 17-mer SEQ ID NO:4 corresponds to residues 11-27 of mature OvIFN.tau., and contains residues non-identical to HuIFN.alpha. at positions 11, 13, 14, 16, 19, 20, 22, 24, and 27. In another embodiment, the method comprises substituting the sequence of HuIFN.alpha. between residues 6-27 with a 22-mer defined by SEQ ID NO:6. In particular, the sequence of mature HuIFN.alpha. between residues 6-27 is SEQ ID NO:5. The 22-mer SEQ ID NO:6 corresponds to residues 6-27 of mature OvIFN.tau., and contains residues non-identical to HuIFN.alpha. 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.alpha.. The method includes substituting, for one or more of the amino acids at positions 19, 20, 22, 24, and 27 of mature HuIFN.alpha., an amino acid effective to substantially reduce the specific toxicity of the polypeptide in mononuclear cells in culture, where the mature HuIFN.alpha. sequence between residues 28-166 is substantially unchanged. In one embodiment, said substituting is accomplished by substituting the sequence of HuIFN.alpha. between residues 1-27 with the 27-mer defined by SEQ ID NO:8. In particular, the sequence of mature HuIFN.alpha. between residues 1-27 is SEQ ID NO:7. The 27-mer SEQ ID NO:8 corresponds to residues 1-27 of mature OvIFN.tau., and contains residues non-identical to mature HuIFN.alpha. 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.alpha. analog for use in human therapy. This analog comprises a mature HuIFN.alpha. 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.alpha. residues. The analog is characterized as having a substantially reduced specific toxicity relative to native human IFN.alpha., 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, for the amino acid at position 27. In another embodiment, the substituted amino acid may include a class V amino acid, in particular Leu, for the amino acid at position 24.

In another embodiment, the analog comprises mature human IFN.alpha. substituted between residues 19-27 with the 9-mer of SEQ ID NO:2. In another embodiment, the analog comprises mature human IFN.alpha. substituted between residues 11-27 with the 17-mer of SEQ ID NO:4. In another embodiment, the analog comprises mature human IFN.alpha. substituted between residues 6-27 with the 22-mer of SEQ ID NO:6.

In a related aspect, the invention includes a low-toxicity human IFN.alpha. analog for use in human therapy, comprising a mature human IFN.alpha. protein having, at one or more of the amino acid positions 19, 20, 22, 24, and 27, a substituted amino acid, with the mature human IFN.alpha. sequence between residues 28-166 being substantially unchanged. The analog is characterized by a substantially reduced specific toxicity relative to native mature human IFN.alpha. as evidenced by an increased viability of mononuclear cells in culture. In one embodiment, the analog comprises mature human IFN.alpha. substituted between residues 1-27 with the 27-mer of SEQ ID NO:8.

The invention further includes a method of inhibiting tumor cell growth. In the method, the tumor cells are contacted with a low-toxicity IFN.alpha. analog of the type described above at a concentration effective to inhibit growth of the tumor cells. The low-toxicity IFN.alpha. analog may be a part of any acceptable pharmacological formulation. Tumor cells whose growth may be inhibited by a low-toxicity IFN.alpha. analog include, but are not limited to, carcinoma cells, hematopoietic cancer cells, leukemia cells, lymphoma cells, and melanoma cells. In one embodiment, the tumor cells are steroid-sensitive tumor cells, for example, mammary tumor cells.

In yet another aspect of the present invention, a low-toxicity IFN.alpha. analog of the type described above is used in a method of inhibiting viral replication. In this method, cells infected with a virus are contacted with the low-toxicity IFN.alpha. compound at a concentration effective to inhibit viral replication within said cells. The low-toxicity IFN.alpha. may be a part of any acceptable pharmacological formulation. The replication of both RNA and DNA viruses may be inhibited by low-toxicity human IFN.alpha.. Exemplary RNA viruses include feline leukemia virus, ovine progressive pneumonia virus, ovine lentivirus, equine infectious anemia virus, bovine immunodeficiency virus, visna-maedi virus, caprine arthritis encephalitis virus, human immunodeficiency virus (HIV) or hepatitis c virus (HCV). An exemplary DNA virus is hepatitis B virus (HBV).

In still another aspect, the present invention includes a method of treating an autoimmune disease in a subject in need of such treatment. In one embodiment, the autoimmune disease is multiple sclerosis. The method includes administering, to the subject, a pharmaceutically effective amount of a low-toxicity human IFN.alpha. analog of the type described above.

In another aspect, the present invention includes a method of treating chronic inflammation in a subject in need of such treatment. In one embodiment, the chronic inflammation arises from ulcerative colitis. The method includes administering, to the subject, a pharmaceutically effective amount of a low-toxicity human IFN.alpha. analog of the type described above.

In yet another aspect, the invention includes a method of treating any disease condition which is responsive to intravenously-administered IFN.alpha., by orally administering a low-toxicity human IFN.alpha. analog of the type described above. Orally-administered analog is preferably ingested by the subject.

Claim 1 of 42 Claims

It is claimed:

1. A polypeptide analog of a native human interferon (IFN)-.alpha. protein,

wherein the sequence of amino acids in the analog that corresponds to the sequence of residues 1-27 of the native IFN-.alpha. protein differs therefrom at one or more of positions 19, 20, 22, 24, and 27, said sequences differing only at said positions, provided that said sequence in the analog does not differ from said corresponding sequence in the native IFN-.alpha. only by the presence of Ser, Thr, Asn, Gln, or Gly at the amino acid residue corresponding to position 22;

and wherein the analog is capable of exhibiting lower toxicity relative to the native IFN-.alpha. in an assay comprising:

(a) incubating a first sample of PBMCs for seven days in a culture medium comprising at least 2000 antiviral units/ml of the analog;

(b) incubating a second sample of PBMCs for seven days in a culture medium having an equal concentration, as antiviral units/ml, of the native IFN-.alpha.; and

(c) comparing the percentage of viable cells remaining in the first sample with that in the second sample, whereby a higher percentage of viable cells indicates the relatively lower toxicity of the IFN species in the culture medium.

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