Granulysin, method of obtaining same, and uses
The present invention relates to granulysin, method of obtaining same, and uses, specifically to the granulysin polypeptide for the use thereof as a medicinal product via the systemic route and to a chimeric molecule comprising a recombinant antibody targeting a tumor antigen and the granulysin polypeptide.
1. A pharmaceutical composition comprising a molecule comprising:
a) a recombinant antibody targeting a tumor antigen, wherein the recombinant antibody comprises anti-CEA scFv MFE23 of SEQ ID No: 2; and
b) a granulysin polypeptide comprising SEQ ID NO:1,
wherein said recombinant antibody and said granulysin polypeptide are part of a single polypeptide chain; and
wherein the recombinant antibody is located at an N-terminal end of the single polypeptide chain and is physically bound by a peptide connector to the granulysin polypeptide.
2. The pharmaceutical composition according to claim 1 , wherein said peptide connector has a length of 5 to 40 amino acids.
3. The pharmaceutical composition according to claim 1 , wherein said peptide connector comprises 2 or more amino acids selected from the group consisting of Gly, Ser, Ala, and Thr.
4. The pharmaceutical composition according to claim 1 , wherein said peptide connector comprises SEQ ID NO: 3.
5. The pharmaceutical composition according to claim 1 , wherein said recombinant antibody consists of anti-CEA scFv MFE23 of SEQ ID NO: 2, wherein said granulysin polypeptide consists of the sequence SEQ ID NO: 1, and wherein said peptide connector consists of the sequence SEQ ID NO: 3.
6. The pharmaceutical composition according to claim 1 , wherein said molecule comprises or consists of the polypeptide of sequence SEQ ID NO: 4.
7. The pharmaceutical composition of claim 1 , wherein the peptide connector has a length of 10 to 30 amino acids.
8. The pharmaceutical composition of claim 1 , wherein the peptide connector has a length of about 20 amino acids.
9. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is formulated for systemic administration.
10. The pharmaceutical composition of claim 1 , wherein the recombinant molecule is expressed in Pichia pastoris.
11. The pharmaceutical composition of claim 1 , wherein the granulysin polypeptide is bound to a polyhistidine sequence.
12. A nucleic acid sequence encoding said molecule of claim 1 .
13. The nucleic acid sequence according to claim 12 , wherein said nucleic acid sequence further comprises a sequence encoding a signal peptide.
14. The nucleic acid sequence of claim 13 , wherein said signal peptide is a factor alpha.
15. A recombinant expression vector comprising said a nucleic acid sequence according to claim 12 .
16. A host cell comprising the recombinant expression vector according to claim 15 .
17. A production process for producing the molecule according to claim 1 , comprising the steps of:
a) introducing a recombinant expression vector comprising a nucleic acid sequence encoding the molecule in a host cell; and
b) culturing the host cell under conditions which allow expression of the nucleic acid sequence to obtain an expressed polypeptide, and
c) optionally isolating or purifying, or both, the expressed polypeptide.
18. The production process according to claim 17 , wherein step b) is performed at a pH between 4.9 and 5.2 and at a temperature between 15° C. and 21° C.
19. The method of claim 18 , wherein step b) is performed at pH 5 and at a temperature of 18° C.
20. The method of claim 17 , wherein the nucleic acid sequence further comprises a sequence encoding a signal peptide, wherein the signal peptide is a factor alpha.
21. A method of treating hematological or solid tumors, the method comprising administering the pharmaceutical composition of claim 1 to a patient in need thereof.