Expression and export of interferon-alpha proteins as Fc fusion proteins
Disclosed are nucleic acid sequences, for example, DNA or RNA sequences, which encode an immunoglobulin Fc-Interferon-alpha fusion protein. The nucleic acid sequences can be inserted into a suitable expression vector and expressed in mammalian cells. Also disclosed is a family of immunoglobulin Fc-Interferon-alpha fusion proteins that can be produced by expression of such nucleic acid sequences. Also disclosed are methods of using such nucleic acid sequences and/or fusion proteins for treating conditions, for example, hepatitis, which are alleviated by the administration of interferon-alpha.
1 . A nucleic acid molecule encoding a fusion protein comprising:
(a) a signal sequence;
(b) an immunoglobulin Fc region; and
(c) a target protein sequence comprising interferon-alpha,
wherein the signal sequence, the immunoglobulin Fc region and the target protein sequence are encoded serially in a 5′ to 3′ direction.
2 . The nucleic acid of claim 1 wherein the immunoglobulin Fc region comprises an immunoglobulin hinge region.
3 . The nucleic acid of claim 1 wherein the immunoglobulin Fc region comprises an immunoglobulin hinge region and an immunoglobulin heavy chain constant region domain.
4 . The nucleic acid of claim 1 wherein the immunoglobulin Fc region comprises an immunoglobulin hinge region and an immunoglobulin CH3 domain.
5 . The nucleic acid of claim 1 , wherein the immunoglobulin Fc region comprises a hinge region, a CH2 domain and a CH3 domain.
6 . The nucleic acid of claim 5 wherein the immunoglobulin Fc region comprises a portion of an immunoglobulin gamma sequence.
7 . The nucleic acid of claim 6 wherein the immunoglobulin gamma is human immunoglobulin gamma1.
8 . A replicable expression vector for transfecting a mammalian cell, the vector comprising the nucleic acid of claim 1 .
9 . The replicable expression vector of claim 8 wherein the vector is a viral vector.
10 . A mammalian cell harboring the nucleic acid of claim 1 .
11 . A fusion protein comprising in an amino terminal to carboxy terminal direction an immunoglobulin Fc region and a target protein comprising interferon-alpha.
12 . The fusion protein of claim 11 wherein the interferon-alpha comprises an amino acid sequence set forth in SEQ. ID. NO.: 2, 7 or 8-21 or a species or allelic variant thereof.
13 . The fusion protein of claim 11 wherein the target protein comprises at least two interferon-alpha molecules linked by a polypeptide linker.
14 . The fusion protein of claim 13 further comprising a polypeptide linker linking the immunoglobulin Fc region to the target protein.
15 . The fusion protein of claim 11 wherein the immunoglobulin Fc region comprises an immunoglobulin hinge region and an immunoglobulin heavy chain constant region domain.
16 . The fusion protein of claim 15 , wherein the heavy chain constant region domain comprises a CH3 domain.
17 . The fusion protein of claim 11 wherein the immunoglobulin Fc region comprises a hinge region, a CH2 domain and a CH3 domain.
18 . A multimeric protein comprising at least two fusion proteins of claim 11 linked via a covalent bond.
19 . The protein of claim 18 wherein the covalent bond is a disulfide bond.
20 . A method of producing a fusion protein comprising the steps of:
(a) providing the mammalian cell of claim 10; and
(b) culturing the mammalian cell to produce the fusion protein.
21 . The method of claim 20 comprising the additional step of collecting the fusion protein.
22 . The method of claim 20 comprising the additional step of purifying the fusion protein.
23 . The method of claim 20 comprising the additional step of cleaving with a proteolytic enzyme the immunoglobulin Fc region from the target protein at a proteolytic cleavage site disposed between the immunoglobulin Fc region and the target protein.
24 . A method of treating a condition alleviated by the administration of interferon-alpha comprising the step of administering the nucleic acid of claim 1 to a mammal having the condition.
25 . A method of treating a condition alleviated by the administration of interferon-alpha comprising the step of administering the vector of claim 8 to a mammal having the condition.
26 . A method of treating a condition alleviated by the administration of interferon-alpha comprising the step of administering the fusion protein of claim 11 to a mammal having the condition.
27 . A method of treating a condition alleviated by the administration of interferon-alpha comprising the step of administering protein of claim 18 to a mammal having the condition.
28 . The method of claim 26 wherein the condition is a liver disorder.
29 . The method of claim 28 wherein the liver disorder is hepatitis.