IL-7 fusion proteins
The invention is directed to a fusion protein which includes a first portion including an immunoglobulin (Ig) chain and a second portion including interleukin-7 (IL-7).
1. A method of preparing a fusion protein comprising:
expressing in a host cell a nucleic acid encoding a fusion protein comprising
a first portion comprising an Fc region from IgG1 or IgG2, wherein the Fc region comprises a hinge region, a CH2 domain, and a CH3 domain, and
a second portion comprising a polypeptide having an amino acid sequence comprising at least 95% sequence identity with wild-type mature human interleukin-7 (IL-7), wherein wild-type human IL-7 consists of amino acid residues 26-177 of SEQ ID NO:1; and
harvesting the fusion protein, wherein the amino acid residues corresponding to positions 70 and 91 of wild-type mature human IL-7 are glycosylated and the amino acid residue corresponding to position 116 of IL-7 is non-glycosylated.
2. The method of claim 1 , wherein the fusion protein comprises a linker between the first portion and the second portion.
3. The method of claim 1 , wherein the nucleic acid encodes for an asparagine residue corresponding to position 116 of IL-7.
4. The method of claim 1 , wherein the Fc region is from IgG1.
5. The method of claim 4 , wherein the Fc region comprises an amino acid substitution at position Asn297.
6. The method of claim 5 , wherein the Fc region comprises an amino acid substitution at position Tyr296.
7. The method of claim 6 , wherein the amino acid substitutions are Asn297Gln and Tyr296Ala.
8. The method of claim 1 , wherein the Fc region is from IgG2.
9. The method of claim 8 , wherein the Fc region further comprises an IgG1 hinge.
10. The method of claim 8 , wherein the Fc region comprises an amino acid substitution in place of Asn at the glycosylation site of the Fc region.
11. The method of claim 10 , wherein the Fc region further comprises an amino acid substitution in place of the Phe residue adjacent the Asn residue at the glycosylation site of the Fc region.
12. The method of claim 11 , wherein Ala is substituted in place of Phe and Gln is substituted in place of Asn at the glycosylation site of the Fc region.
13. The method of claim 1 , wherein the host cell is a mouse myeloma NS/0 cell.
14. The method of claim 1 , wherein the second portion comprises wild-type mature IL-7 consisting of amino acid residues 26-177 of SEQ ID NO:1.
15. A method of preparing a fusion protein comprising:
expressing in a host cell a nucleic acid encoding a fusion protein comprising
a first portion comprising an Fc region from IgG1 or IgG2, wherein the Fc region comprises a hinge region, a CH2 domain, and a CH3 domain, and
a second portion comprising a polypeptide having an amino acid sequence comprising at least 95% sequence identity with wild-type mature human interleukin-7 (IL-7), wherein wild-type human IL-7 consists of amino acid residues 26-177 of SEQ ID NO:1, except that residues 121 to 138 of SEQ ID NO:1 are deleted in said second portion; and
harvesting the fusion protein, wherein the amino acid residues corresponding to positions 70 and 91 of wild-type mature human IL-7 are glycosylated and the amino acid residue corresponding to position 116 of IL-7 is non-glycosylated.
16. The method of claim 15 , wherein the Fc region is from IgG1.
17. The method of claim 16 , wherein the Fc region comprises the amino acid substitutions Asn297Gln and Tyr296Ala.
18. The method of claim 15 , wherein the Fc region is from IgG2.
19. The method of claim 18 , wherein the Fc region further comprises an amino acid substitution of Ala in place of the Phe residue adjacent the Asn residue at the glycosylation site of the Fc region, and an amino acid substitution of Gln in place of the Asn residue at the glycosylation site of the Fc region.