Use of peptidylglycine alpha-amidating monooxigenase (pam) for c-terminal amidation

Patent metadata

Field	Value
Publication number	US-2016333386-A1
Application number	US-201615185477-A
Country	US
Kind code	A1
Filing date	Jun 17, 2016
Priority date	Dec 20, 2013
Publication date	Nov 17, 2016
Grant date	—

Abstract

One aspect as reported herein is a method for in vivo C-terminal amidation of a polypeptide characterized in that both the polypeptide (to be amidated) and human peptidylglycine alpha-amidating monooxigenase (PAM) are recombinantly co-expressed in a mammalian cell.

First claim

1 . A method for in vivo C-terminal amidation of a polypeptide characterized in that both the polypeptide and human peptidylglycine alpha-amidating monooxigenase (PAM) are recombinantly co-expressed in a mammalian cell.

2 . A method for the recombinant production of a C-terminally amidated polypeptide characterized in that both the polypeptide and human peptidylglycine alpha-amidating monooxigenase (PAM) are recombinantly co-expressed in a mammalian cell.

3 . The method according to any of the preceding claims, characterized in that the human peptidylglycine alpha-amidating monooxigenase (PAM) is a PAM 3 (SEQ ID NO: 02).

4 . The method according to any of the preceding claims, characterized in that the mammalian cell comprises a first nucleic acid encoding the polypeptide and a second nucleic acid encoding the PAM.

5 . The method according to claim 4 , characterized in that the ratio of the first nucleic acid to the second nucleic acid is from about 90:10 to about 40:60.

6 . The method according claim 4 , characterized in that the ratio of the first nucleic acid to the second nucleic acid is from about 70:30 to about 60:40.

7 . The method according to any of the preceding claims, characterized in that the polypeptide is fused to the C-Terminus of an antibody heavy chain or the Fc region thereof.

8 . The method according any of the preceding claims, characterized in that the polypeptide is Neurokinin, Allatostatin, Lem-KI, TRH, Red Pigment Concentrating Hormone, Calcitonin, CRF, LHRH, Leucopyrokinin, Gastrin I, Pigment Dispersing Hormone, Dermorphin, Oxytocin, Substance P, NPY, FMRFamide, Bombesin, Amylin, [Arg 8 ]Vasopressin, BId-GrTH, Calcitonin, Cam-HrTH-II, Gastrin Releasing Peptide, Neuromedin B, Pancreastatin, Conotoxin M1, Secretin, GHRF, Melittin, Sarcotoxin 1A, VIP, α-MSH or MIF-1.

9 . The method according any of the preceding claims, characterized in that the polypeptide is peptide YY (PYY 3-36) of SEQ ID NO: 05.

10 . Use of a human peptidylglycine alpha-amidating monooxigenase (PAM) for the recombinant production of a C-terminally amidated polypeptide, characterized in that both the polypeptide and the human PAM are recombinantly co-expressed in a mammalian cell.

Assignees

• Hoffmann La Roche

Inventors

• Hoffmann Eike
• Tiefenthaler Georg

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12Y114/17003

  Peptidylglycine monooxygenase (1.14.17.3) · CPC title

• C12Y403/02005

  Peptidylamidoglycolate lyase (4.3.2.5) · CPC title

• C07K14/575

  Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695) · CPC title

• C07K1/003 Primary

  by transforming the C-terminal amino acid to amides · CPC title