US 11,959,913 B2
Quantitative cellular method for determining the biological activity of an anti-CD26 ligand
Antonio Francesco Di Naro, Morcote (CH)
Assigned to ADIENNE S.A., Lugano (CH)
Appl. No. 16/768,206
Filed by ADIENNE S.A., Lugano (CH)
PCT Filed Dec. 21, 2018, PCT No. PCT/IB2018/060507
§ 371(c)(1), (2) Date May 29, 2020,
PCT Pub. No. WO2019/123410, PCT Pub. Date Jun. 27, 2019.
Claims priority of application No. 102017000148959 (IT), filed on Dec. 22, 2017.
Prior Publication US 2020/0319169 A1, Oct. 8, 2020
Int. Cl. G01N 33/533 (2006.01); C07K 16/28 (2006.01); G01N 33/50 (2006.01); G06F 17/18 (2006.01); C07K 16/40 (2006.01)
CPC G01N 33/533 (2013.01) [G01N 33/505 (2013.01); G06F 17/18 (2013.01); C07K 16/2896 (2013.01); C07K 16/40 (2013.01); G01N 2333/70596 (2013.01)] 12 Claims
 
1. A method for the in vitro determination of the potency of an anti-CD26 ligand comprising the following steps:
a) incubating at 37° C. or at room temperature a population of human T lymphocytes expressing the CD26 receptor in a percentage higher than 75% with an anti-CD26 ligand at a concentration ranging from 0.001 μg/ml to 150 μg/ml;
b) contacting said human T lymphocytes with an anti-CD26 antibody conjugated to a fluorochrome; wherein the anti-CD26 antibody recognizes a different CD26 epitope from the epitope recognized by the anti-CD26 ligand used in step a);
c) determining the mean fluorescence intensity (MFI) of CD26 measured for the sample of cells treated with the anti-CD26 ligand (MFIT) and the MFI value of untreated cells (MFINT) by means of cytofluorimetric analysis;
d) evaluating the internalization percentage of the CD26 receptor (% int CD26) or RFI calculated according to the following formula:

OG Complex Work Unit Math
wherein if the value of % intCD26 is:
less than 20% it indicates a low potency of the anti-CD26 ligand;
ranging from 20% to 30% it indicates a medium potency of the anti-CD26 ligand; and
higher than 30% indicates a high potency of the anti-CD26 ligand.