Quantitative cellular method for determining the biological activity of an anti-CD26 ligand

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 (MFI T ) and the MFI value of untreated cells (MFI NT ) by means of cytofluorimetric analysis;

d) evaluating the internalization percentage of the CD26 receptor (% int CD26) or RFI calculated according to the following formula:

%

⁢

int

⁢

C

⁢

D

⁢

26

=

100

-

(

M

⁢

F

⁢

I

T

M

⁢

F

⁢

I

NT

×

100

)

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.

2. The method according to claim 1 , wherein said concentration of the anti-CD26 ligand of step a) ranges from 0.01 μg/ml to 100 μg/ml.

3. The method according to claim 1 , wherein said fluorochrome of step b) is selected from the group consisting of fluorescein isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE), PE-Cy7, APC-H7, peridinin-chlorophyll-protein (PerCP) and PE-Cy5.5.

4. The method according to claim 1 , wherein the population of human CD26+ T lymphocytes of step a) is selected from a population of primary T lymphocytes and a tumoral cell line of human T lymphocytes.

5. The method according to claim 4 , wherein said tumoral cell line of human T lymphocytes is the Karpas 299 cell line.

6. The method according to claim 1 , wherein the cytofluorimetric analysis of step c) is carried out by fluorescence-activated cell sorting (FACS).

7. The method according to claim 1 , further comprising assaying for the inhibition of inflammatory cytokine production; wherein the inflammatory cytokines are selected from the group consisting of IL-8, IL-β3, IL-6, IL-2, GM-CSF, IL-6 and TNF-α; and wherein the assay is performed on the population of human CD26+T lymphocytes of step a).

8. The method according to claim 7 , wherein said inhibition of the production of cytokines is evaluated by the MesoScale Discovery assay.

9. The method according to claim 7 , wherein said population of human CD26+T lymphocytes is the Karpas 299 cell line.

10. The method according to claim 2 , wherein the concentration of anti-CD26 ligand is from 0.01 μg/ml to 2 μg/ml.

11. The method according to claim 2 , wherein the concentration of anti-CD26 ligand is from 0.01 μg/ml to 0.5 μg/ml.

12. The method according to claim 3 , wherein the fluorochrome is APC.