JPWO2020092975A5

JPWO2020092975A5 -

Info

Publication number: JPWO2020092975A5
Application number: JP2021523351A
Authority: JP
Publication date: 2022-11-04

Claims

A method of assaying antigen-specific cytotoxicity of T lymphocytes (T cells) in a microfluidic device, comprising:
placing the T cells in the microfluidic device;
placing target cells in proximity to said T cells;
determining the viability of the target cells;
including
said microfluidic device comprising a flow region for containing a flow of a first fluid medium and a chamber open to said flow region;
Method.

2. The method of claim 1 , wherein said target cell expresses an antigen to which said T cell is specific.

The assay comprises placing a first capture entity in the vicinity of the T cell configured to capture a first secreted biomolecule of the T cell; and 3. The method of claim 1 or 2 , further comprising detecting the first secreted biomolecule.

4. The method of claim 3 , wherein said first secreted biomolecule is a protein.

5. The method of claim 3 or 4 , wherein said first secreted biomolecule of said T cell is a cytokine.

The cytokine is tumor necrosis factor alpha (TNFα), transforming growth factor beta (TGFβ), interferon gamma (IFNγ), interleukin-1β (IL1β), interleukin-2 (IL2), interleukin-4 (IL4) , interleukin-5 (IL5), interleukin-6 (IL6), interleukin-10 (IL10), interleukin-12 (IL12), interleukin-13 (IL13), interleukin-17A (IL17A), or 6. The method of claim 5 , which is interleukin-22 (IL22).

7. The method of any one of claims 1-6 , wherein the target cells are cancer cells.

8. The method of claim 7 , wherein the target cells express antigens associated with melanoma, breast cancer, or lung cancer.

9. The method of any one of claims 1-8 , wherein said T cells are mammalian T cells.

10. The method of any one of claims 1-9, wherein said T cells are antigen -specific for tumor-associated antigens and/or express chimeric antigen receptors .

10. The method of any one of claims 1-9, wherein said T cells do not express chimeric antigen receptors.

said chamber comprising an isolation enclosure ;
said isolation enclosure comprising a separation region for containing a second fluid medium and a connection region fluidly connecting said separation region to said flow region;
the separation region comprises one opening and is a non-swept region of the microfluidic device;
12. A method according to any one of claims 1-11.

13. The method of any one of claims 1-12, wherein the T cells and the target cells are each arranged in the chamber.

13. The method of claim 12 , wherein said T cells and said target cells are each arranged in said isolation region of said isolation pen.

15. The method of any one of claims 1-14, wherein determining the viability of the target cells comprises contacting the target cells with a detectable marker configured to label non-viable cells. described method.

16. The method of claim 15 , wherein the detectable marker configured to label non-viable cells is configured to label apoptotic cells.

16. The method of claim 15 , wherein the detectable marker configured to label non-viable cells is configured to label calcium flux or mitochondrial membrane potential.

18. The method of any one of claims 1-17, wherein determining the viability of the target cells is repeated over multiple periods of exposure to the T cells.

19. Any one of claims 1-18 , further comprising labeling said T cells for the presence of one or more cell surface markers associated with proliferation, activation, metabolic activity, memory, exhaustion, and/or maturation. The method described in .

A kit for assaying antigen-specific cytotoxicity by T lymphocytes (T cells) in a microfluidic device, comprising:
a microfluidic device comprising a flow region for containing a flow of a first fluid medium and a chamber open to said flow region;
a cytotoxicity detection reagent configured to detect target cell viability ;
Kit with .

21. The kit of Claim 20 , wherein said cytotoxicity detection reagent comprises a reagent configured to label apoptotic cells.

21. The kit of claim 20 , wherein said cytotoxicity detection reagents comprise reagents configured to detect calcium flux or mitochondrial membrane potential.

23. The kit of any one of claims 20-22 , further comprising a first capture entity configured to capture a first secreted biomolecule of T cells.

said chamber comprising an isolation enclosure;
said isolation enclosure comprising a separation region for containing a second fluid medium and a connection region fluidly connecting said separation region to said flow region;
the separation region comprises one opening and is a non-swept region of the microfluidic device;
24. A kit according to any one of claims 20-23.