Methods for selection and stimulation of cells and apparatus for same

A method of on-column stimulation of T cells, the method comprising:

• (a) adding a sample comprising a plurality of T cells to a stationary phase comprised in a chromatography column, said stationary phase comprising a selection agent that binds to a selection marker on the surface of one or more of the plurality of T cells, thereby immobilizing the one or more of the plurality of T cells on the stationary phase;
• (b) adding, to the stationary phase, a stimulatory reagent comprising one or more stimulatory agents capable of delivering a stimulatory signal in one or more of said plurality of T cells, thereby initiating incubation of the stimulatory reagent with the one or more of said plurality of T cells;
• and
• (c) within 24 hours of the initiating incubation, collecting one or more of said plurality of T cells detached from the stationary phase by downregulation of the selection marker and by gravity flow without the addition of a competition agent or free binding agent to elute the plurality of T cells from the stationary phase, thereby generating a composition comprising stimulated T cells.

A method of on-column stimulation of T cells, comprising:

• (a) combining (i) a sample comprising a plurality of T cells and (ii) a stationary phase comprised in a chromatography column and comprising a selection agent capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, wherein specific binding of the selection agent to a selection marker immobilizes said one or more of the plurality of T cells on the stationary phase;
• (b) adding, to the stationary phase, a stimulatory reagent comprising one or more stimulatory agents capable of delivering a stimulatory signal in T cells, thereby initiating incubation of the stimulatory reagent with the one or more of the plurality of T cells;
• and
• (c) within 24 hours of the initiating incubation, collecting one or more of said plurality of T cells detached from the stationary phase by downregulation of the selection marker and by gravity flow without the addition of a competition agent or free binding agent to elute the plurality of T cells from the stationary phase, thereby generating a composition comprising stimulated T cells.

A method of on-column stimulation of T cells, the method comprising adding an oligomeric stimulatory reagent to a stationary phase comprising a plurality of T cells immobilized on the stationary phase, thereby initiating incubation of the stimulatory reagent with one or more T cells of the plurality of T cells, wherein:

• the stationary phase is comprised in a chromatography column and comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells, wherein specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes said one or more T cells on the stationary phase;
• and
• the oligomeric stimulatory reagent comprises (i) a plurality of streptavidin or streptavidin mutein molecules and (ii) one or more stimulatory agents capable of delivering a stimulatory signal in one or more T cells, wherein the size of the oligomeric stimulatory reagent comprises (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5×106 g/mol;
• and/or (iii) at least 100 streptavidin or streptavidin mutein tetramers;
• and
• within 24 hours of the initiating incubation, collecting one or more of the plurality of T cells detached from the stationary phase by downregulation of the selection marker and by gravity flow without the addition of a competition agent or free binding agent to elute the plurality of T cells from the stationary phase, thereby generating a composition containing stimulated T cells.

A method of on-column stimulation of T cells, the method comprising:

• (a) adding an oligomeric stimulatory reagent capable of delivering a stimulatory signal in T cells to a stationary phase comprising a plurality of T cells immobilized on the stationary phase, thereby initiating incubation of the stimulatory reagent with one or more T cells, wherein:
• the stationary phase is comprised in a chromatography column and comprises a selection agent capable of specifically binding to a selection marker on the surface of one or more T cells or a subset thereof, wherein specific binding of the selection agent to a selection marker expressed by the one or more T cells or a subset thereof immobilizes said plurality of T cells on the stationary phase, and wherein the selection agent is a Fab fragment capable of specifically binding to a selection marker selected from the group consisting of CD3, CD4, and CD8;
• and
• the oligomeric stimulatory reagent comprises (i) a plurality of streptavidin mutein molecules, (ii) a first stimulatory agent capable of delivering a stimulatory signal in one or more T cells, wherein the first stimulatory agent is a Fab fragment capable of specifically binding to CD3, and (iii) a second stimulatory agent capable of enhancing, dampening, or modifying the stimulatory signal, wherein the second stimulatory agent is a Fab fragment capable of specifically binding to CD28, and wherein the size of the oligomeric stimulatory reagent comprises (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5×106 g/mol;
• and/or (iii) at least 100 streptavidin or streptavidin mutein tetramers;
• and
• (b) within 24 hours of initiating incubation, collecting one or more of the plurality of T cells detached from the stationary phase by downregulation of the selection marker and by gravity flow without the addition of a competition agent or free binding agent to elute the plurality of T cells from the stationary phase.