Dimeric alpha interferon PEGylated site-specifically shows enhanced and prolonged efficacy in vivo
The present invention concerns methods and compositions for forming PEGylated complexes of defined stoichiometry and structure. In preferred embodiments, the PEGylated complex is formed using dock-and-lock technology, by attaching a therapeutic agent to a DDD sequence and attaching a PEG moiety to an AD sequence and allowing the DDD sequence to bind to the AD sequence in a 2:1 stoichiometry, to form PEGylated complexes with two therapeutic agents and one PEG moiety. In alternative embodiments, the therapeutic agent may be attached to the AD sequence and the PEG to the DDD sequence to form PEGylated complexes with two PEG moieties and one therapeutic agent. In more preferred embodiments, the therapeutic agent may comprise any peptide or protein of physiologic or therapeutic activity, preferably a cytokine, more preferably interferon-α2b. The PEGylated complexes exhibit a significantly slower rate of clearance when injected into a subject and are of use for treatment of a wide variety of diseases.
1. A method of treating cancer or a viral infection comprising:
a) obtaining a PEGylated interferon-α (IFN-α) or interferon-β (IFN-β) complex comprising;
i) a PEG (polyethylene glycol) moiety covalently attached to a first peptide;
ii) a fusion protein comprising IFN-α or IFN-β and a second peptide; and
b) administering the PEGylated IFN-α or IFN-β complex to a subject with cancer, a viral infection or autoimmune disease;
wherein two copies of the second peptide form a dimer that binds to the first peptide to form a PEGylated complex, wherein the amino acid sequence of the second peptide is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, and the amino acid sequence of the first peptide is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.
2. The method of claim 1 , wherein the second peptide is attached to the C-terminal end of IFN-α or IFN-β.
3. The method of claim 1 , wherein the IFN-α is IFN-α 2b.
4. The method of claim 1 , wherein the first peptide, or the first peptide covalently attached to the PEG moiety, is selected from the group consisting of:
IMP350
(SEQ ID NO: 21)
CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH 2 ;
IMP360
(SEQ ID NO: 22)
CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-G-EDANS;
IMP362
(SEQ ID NO: 22)
PEG 20k -CO-CGQIEYLAKQIVDNAIQQAGCG-NH-(CH 2 ) 2 -EDANS;
IMP413
(SEQ ID NO: 22)
mPEG 30K -CGQIEYLAKQIVDNAIQQAGCG-NH-(CH 2 ) 2 -EDANS;
IMP421
(SEQ ID NO: 22)
Ac-C-PEG 3 -C(SStBu)GQIEYLAKQIVDNAIQQAGCG-NH 2
and
IMP457
(SEQ ID NO: 22)
Ac-C(mPEG2-Suc 40K)-PEG 3 -CGQIEYLAKQIVDNAIQQAGCG-
NH 2 .
5. The method of claim 1 , wherein the PEGylated complex has a higher anti-viral specific activity than Peginterferon alfa-2b and Peginterferon alfa-2a.
6. The method of claim 1 , wherein the PEGylated complex has a higher anti-proliferative effect on cancer cells in vitro than Peginterferon alfa-2b.
7. The method of claim 1 , wherein the clearance rate of the PEGylated complex from serum is at least an order of magnitude slower than the clearance rate of the unPEGylated IFN -α.
8. The method of claim 1 , wherein the PEGylated complex has greater anti-tumor efficacy in vivo than Peginterferon alfa-2b.
9. The method of claim 1 , wherein the PEG moiety is capped at one end with a methoxy group.
10. The method of claim 1 , wherein the viral infection is hepatitis B, hepatitis C, dengue virus, influenza virus, rhinovirus, cytomegalovirus, herpes simplex, vaccinia virus or encephalomyocarditis virus.