Combined invasion and cytotoxicity assay using chemokine secreting target cells
Provided herein are compositions and methods for detecting migration of effector cells towards a target cell, and cytotoxicity of the migrated effector cells against the target cells. The effector cells are modified to express a homing or migratory receptor, and the target cells are modified to express the cognate ligand. The methods can be carried out in a Boyden chamber or tranwells with a porous membrane between the wells. The membrane can be coated with an extracellular matrix component to simulate a solid tumor environment.
1 . A method for detecting migration and cytotoxicity of effector cells, the method comprising:
providing a device comprising two or more chambers, wherein at least a first chamber is separated by a membrane from a second chamber, the membrane comprising pores having a diameter that permits cells to pass through the membrane;
adding effector cells to the first chamber, wherein the effector cell is modified to express a homing receptor, wherein the homing receptor is CCR7;
adding target cells to the second chamber, wherein the target cell is modified to express a ligand that binds to the homing receptor;
allowing the effector cells to migrate from the first chamber to the second chamber; and
measuring the number of target cells killed by the effector cells in the second chamber, wherein the effector cells are NK-92 cells.
2 . The method of claim 1 , wherein the first chamber is located above the second chamber.
3 . The method of claim 1 , wherein the membrane comprises an extra-cellular matrix component or analog that inhibits migration of the effector cell.
4 . The method of claim 1 , wherein the effector cell is stained with a vital dye.
5 . The method of claim 1 , wherein the method further comprises determining the number of effector cells that migrated into the second chamber.
6 . The method of claim 1 , wherein the ligand expressed by the target cell is selected from C-C Motif Chemokine Ligand 19 (CCL19 (SEQ ID NO: 2)), C-C Motif Chemokine Ligand 21 (CCL21 (SEQ ID NO: 4)), interleukin 8 (IL8 or CXCL8 (SEQ ID NO: 6)), C-X-C motif chemokine ligand 1 (CXCL1 (SEQ ID NO: 8)), or C-X-C Motif Chemokine Ligand 14 (CXCL14 (SEQ ID NO: 10)).
7 . The method of claim 1 , wherein the amino acid sequence of CCR7 has at least 90% identity to SEQ ID NO: 14, and the effector cell is capable of migrating towards the ligand expressed by the target cell.
8 . The method of claim 1 , wherein the effector cell is further modified to express a chimeric antigen receptor (CAR) and wherein the CAR specifically binds:
i) CD19 and comprises an amino acid sequence selected from SEQ ID NOs: 46, 47, or 63; or
ii) CD20 and comprises an amino acid sequence of SEQ ID NO: 65; or
iii) GD2 and comprises an amino acid sequence selected from SEQ ID NOs: 80 or 81; or
iv) HER-2 and comprises an amino acid sequence of SEQ ID NO: 77; or
v) CD30 and comprises an amino acid sequence of SEQ ID NO: 75; or
vi) EGFR and comprises an amino acid sequence of SEQ ID NO: 71; or
vii) FAP and comprises an amino acid sequence selected from SEQ ID NOs: 95 or 96; or
viii) CD33 and comprises an amino acid sequence of SEQ ID NO: 67; or
ix) CD123 and comprises an amino acid sequence of SEQ ID NO: 86; or
x) PD-L1 and comprises an amino acid sequence of SEQ ID NO: 88); or
xi) IGF1R and comprises an amino acid sequence of SEQ ID NO: 73; or
xii) CSPG4 and comprises an amino acid sequence of SEQ ID NO: 69; or
xiii) B7-H4 and comprises an amino acid sequence of SEQ ID NO: 90.
9 . The method of claim 8 , wherein the CAR specifically binds CD19, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 46, 47, or 63, and the target cell is killed by the effector cell.
10 . The method of claim 8 , wherein the CAR comprises an Fc Epsilon signaling domain.
11 . The method of claim 1 , wherein the effector cell comprises a nucleic acid encoding a chemokine receptor operably linked to a promoter.
12 . The method of claim 1 , wherein the target cell comprises a nucleic acid encoding a chemokine operably linked to a promoter.
13 . The method of claim 1 , wherein the ligand expressed by the target cell is a ligand that binds CD62L (SEQ ID NO: 12).