IP Library › Granted Patent US 12,729,369

Granted Patent B2

US 12,729,369 · App. 17/635,755 · Granted Sep 8, 2026

Process for manufacturing of antigen-specific T lymphocytes

Inventors: Piotr Trzonkowski (Gdańsk, PL); Dorota Iwaszkiewicz-Grześ (Gdańsk, PL); Mateusz Gliwiński (Kwidzyn, PL)

Assignee: GDANSKI UNIWERSYTET MEDYCZNY

C12N5/0636 C12N5/0637 C12N5/0638 C12N13/00 C12N2500/02 C12N2501/33 C12N2501/998 C12N2502/1157

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Quick Facts

Patent No.: US 12,729,369
App. No.: 17/635,755
Granted: Sep 8, 2026
Kind: B2

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USPTO Patent Center↗ Full Patent PDF↗

Abstract

The invention relates to a new process for manufacturing in vitro antigen-specific T lymphocytes (CellTrAg), marked with intracellular dye and expanded in the presence of monocytes loaded with the antigen and subsequently sorted based on the low intensity of intracellular dye where the low intensity of fluorescence is a marker of antigen-specificity in that a loss of fluorescence correlated with the intensity of proliferation. The antigen specificity is assessed in functional tests in which antigen-specific lymphocytes sorted on the basis of low fluorescence are more active than non-specific lymphocytes with high fluorescence during sort; activity is defined in the case of regulatory T lymphocytes as inhibition of effector lymphocyte function and in the case of T effector lymphocytes as enhancing the characteristics of these cells such as proliferation, production of cytokines and cytotoxic factors.

Claims (13)

1 . A process for ex-vivo expansion of antigen-specific CD4 + CD25highCD127 − /low T regulatory lymphocytes comprising the steps of:

a) Suspend CD4 + CD25highCD127 − /low T regulatory lymphocytes (Tregs) in PBS containing a fluorescent dye to produce intracellularly stained Tregs;

b) Incubate the stained Tregs in the dark;

c) Wash the stained Tregs several times with culture medium;

d) Suspend the stained Tregs in culture medium and contact with gamma-irradiated autologous CD 14 + monocytes loaded with antigen to produce a co-culture;

e) Incubate the co-culture with anti-CD154 and anti-CD28 antibodies; and

f) Sort the antigen-specific Tregs based on the low intensity of intracellular dye; and the unspecific Tregs based on preserved high fluorescence of the intracellular dye.

2 . The process of claim 1 characterized in that the Tregs are suspended in PBS at a concentration of 1×10 6 cells/ml.

3 . The process of claim 1 characterized in that the Tregs are stained with Violet Blue in the final concentration of 1-5 μM.

4 . The process of claim 1 characterized in that the final monocyte: Tregs ratio is 1:1.

5 . The process of claim 1 characterized in that in step (e) the final concentration of anti-CD154 antibodies is 5 μg/ml and the final concentration of anti-CD28 antibodies is 5 μg/ml.

6 . The process of claim 1 further comprising the step of:

a) Characterizing the specificity of Tregs to antigen using a functional test, wherein the functional test comprises assessing the ability of Tregs to suppress the function of T effector lymphocytes.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 16, 2022

From: TRZONKOWSKI, PIOTR; IWASZKIEWICZ-GRZES, DOROTA; GLIWINSKI, MATEUSZ

To: GDANSKI UNIWERSYTET MEDYCZNY

Reel/Frame 059025/0033 →

Priority Claims (1)

PL 430932 · Aug 22, 2019 · national

Continuity (1)

Related Publication 20220333072A1 · Oct 20, 2022

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