US 12,234,279 B2
Peptide construct having a protease-cleavable linker
Scott Crowe, Babraham (GB); Mike West, Babraham (GB); and Kevin Roberts, Babraham (GB)
Assigned to SORRISO PHARMACEUTICALS, INC., San Diego, CA (US)
Filed by Sorriso Pharmaceuticals, Inc., San Diego, CA (US)
Filed on Mar. 9, 2021, as Appl. No. 17/196,498.
Application 17/196,498 is a continuation of application No. 15/717,174, filed on Sep. 27, 2017, abandoned.
Application 15/717,174 is a continuation of application No. PCT/EP2016/057022, filed on Mar. 31, 2016.
Claims priority of application No. 15162112 (EP), filed on Mar. 31, 2015; application No. 15162114 (EP), filed on Mar. 31, 2015; application No. 15162115 (EP), filed on Mar. 31, 2015; and application No. 16152320 (EP), filed on Jan. 21, 2016.
Prior Publication US 2021/0198345 A1, Jul. 1, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 16/12 (2006.01); C07K 16/24 (2006.01)
CPC C07K 16/1282 (2013.01) [C07K 16/241 (2013.01); C07K 2317/56 (2013.01); C07K 2317/64 (2013.01); C07K 2317/94 (2013.01); C07K 2319/50 (2013.01)] 16 Claims
 
1. A method of making a construct comprising a first VHH and a second VHH connected by a labile peptide linker, wherein:
(i) the labile peptide linker is labile to one or more proteases present in the intestinal tract;
(ii) the labile peptide linker is stable to yeast proteases;
(iii) the labile peptide linker comprises of a polypeptide sequence of the format:
[-(GaS)x—B-(GbS)y—]z
wherein
a is 3 to 5
b is 3 to 5
x is 1 or 2
y is 1 or 2
z is 1 to 3 and
B is K; and
(iv) wherein the construct comprises a first VHH and a second VHH;
(v) wherein
(a) the labile peptide linker is labile to one or more proteases present in the intestinal tract such that greater than 50% by mass of the construct is cleaved into first and second VHHs after 160 minutes after mixing in the Trypsin Protease Assay;
(b) the labile peptide linker is stable to yeast proteases such that no more than 10% by mass of the construct is cleaved into first and second VHHs after producing the construct using the Yeast Expression Protocol; or
(c) the first VHH and the second VHH are resistant to one or more proteases present in the intestinal tract such that at least 70% by mass of the first VHH and at least 70% by mass of the second VHH remain uncleaved after 10 minutes after mixing in the Trypsin Protease Assay;
wherein the method comprises providing a host yeast cell capable of expressing the construct, producing the construct in the host yeast cell, and recovering the construct from the host yeast cell.