Shortened CD95-Fc variant fusion proteins, nucleic acids and host cells thereof
The present invention relates to an isolated fusion protein comprising an extracellular CD95 domain or a functional fragment thereof and an Fc domain or functional fragment thereof, formulations providing such fusion protein in a stable form as well as a method for producing such a fusion protein.
1. A fusion protein comprising (i) at least a functional fragment of an N-terminally truncated extracellular cluster of differentiation 95 (CD95) domain, wherein the functional fragment of the N-terminally truncated extracellular CD95 domain consists of the amino acid sequence of SEQ ID NO: 11 or 12, or a sequence having at least 97% identity to SEQ ID NO: 11 or 12, and (ii) at least an Fc domain or a functional fragment thereof, wherein the Fc domain is a human Fc domain consisting of the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 97% identity to SEQ ID NO: 8.
2. The fusion protein according to claim 1 , wherein the functional fragment of the N-terminally truncated extracellular CD95 domain consists of the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12.
3. The fusion protein according to claim 1 , said fusion protein being free of a signal peptide.
4. The fusion protein according to claim 1 , comprising an N-terminally located signal peptide.
5. The fusion protein according to claim 4 , wherein the N-terminally located signal peptide is a naturally occurring CD95 signal peptide.
6. The fusion protein according to claim 5 , wherein the N-terminally located signal peptide is an artificial signal peptide.
7. The fusion protein according to claim 6 , wherein the N-terminally located signal peptide comprises SEQ ID NO:2 or a sequence having at least 90%, or at least 95% identity to SEQ ID NO:2.
8. The fusion protein according to claim 1 , wherein the functional fragment of the N-terminally truncated extracellular CD95 domain is located N-terminally to the at least an Fc domain or the functional fragment thereof.
9. The fusion protein according to claim 6 , wherein the signal peptide is directly fused to the functional fragment of the N-terminally truncated extracellular CD95 domain.
10. The fusion protein according to claim 1 , wherein the functional fragment of the N-terminally truncated extracellular CD95 domain is directly fused to the N-terminal of the Fc domain or to the functional fragment thereof, wherein the two domains have an overlapping amino acid of serine.
11. The fusion protein according to claim 10 , comprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and a sequence having at least 97% identity to SEQ ID NO:5, or SEQ ID NO:6.
12. The fusion protein of claim 1 , wherein the at least an Fc domain or the functional fragment thereof provides a pl in the range of 4.0-8.5.
13. The fusion protein according to claim 12 , wherein the pl is in the range of 4.5-7.8.
14. The fusion protein according to claim 1 , comprising sialic acids.
15. The fusion protein according to claim 1 , which is N-terminally blocked.
16. The fusion protein according to claim 1 , which is not glycosylated in the CH2 domain of the Fc domain or of the functional fragment thereof.
17. A formulation comprising the fusion protein according to claim 1 .
18. The formulation of claim 17 further comprising:
(a) a phosphate,
(b) a viscosity enhancing agent, and/or
(c) having a pH value in the range of 4-8.
19. The fusion protein according to claim 15 , wherein the N-terminally blocked fusion proteins are blocked by pyro-Glu modification.
20. The formulation of claim 18 , wherein the phosphate is in an amount of 20 mM to 100 mM, and the viscosity enhancing agent is sorbitol in an amount of about 0.1-10 weight %.
21. A nucleic acid molecule encoding the fusion protein according to claim 1 .
22. An isolated host cell comprising a nucleic acid molecule of claim 21 .
23. The method for producing the fusion protein according to claim 1 , comprising recombinantly expressing a nucleic acid molecule encoding the fusion protein.
24. The method of claim 23 , comprising the steps of:
(a) producing the fusion protein according to claim 1 by a fed-batch production process providing a cell harvest, and
(b) isolating the fusion protein from the cell harvest.
25. The method according to claim 23 , wherein step (a) comprises a series of cultivation steps of a given master cell batch until relevant harvest parameters are reached, followed by cell sedimentation and filtration of fusion protein containing supernatant, and/or step (b) comprises capture chromatography, virus inactivation, a series of anion and/or cation chromatography, virus filtration and/or adjustment to a desired final protein concentration.
26. A formulation comprising the nucleic acid molecule according to claim 21 .