Methods for selection and stimulation of cells and apparatus for same
Provided herein are methods for selecting and stimulating a plurality of cells in a sample of cells using column chromatography, and collecting the cells without using additional steps or reagents to facilitate detachment of the cells from the column. In some aspects, the methods provided herein reduce the time needed to generate a population of selected and stimulated cells useful for genetic engineering, and ultimately, cell therapy, compared to existing methods. Also provided are articles of manufacture and apparatus thereof.
1 . 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 that specifically binds 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 the selection marker expressed by the one or more T cells immobilizes the one or more T cells on the stationary phase; and
the oligomeric stimulatory reagent comprises one or more stimulatory agents comprising (i) a first stimulatory agent that is an anti-CD3 antibody, and (ii) a second stimulatory agent that is an anti-CD28 antibody; 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, thereby generating a composition comprising stimulated T cells.
2 . A method of on-column stimulation of T cells, the method comprising:
(a) incubating a plurality of T cells immobilized on a stationary phase with one or more stimulatory agents to deliver a stimulatory signal in one or more T cells of the plurality of T cells, said stationary phase comprised in a chromatography column and comprising a selection agent that specifically binds to a selection marker on the surface of the 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 the one or more T cells on the stationary phase; and
(b) within 24 hours of the initiation of the incubation, collecting the one or more 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.
3 . The method of claim 2 , wherein the stationary phase comprises or is immobilized with at least one of the one or more stimulatory agents capable of delivering a stimulatory signal in the one or more T cells.
4 . The method of claim 2 , wherein the method comprises prior to the incubating, adding a stimulatory reagent to the stationary phase, said stimulatory reagent comprising at least one of the one or more stimulatory agents.
5 . 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.
6 . 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.
7 . 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 5×10 6 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.
8 . The method of claim 1 , wherein the collecting one or more of the plurality of T cells from the stationary phase occurs within about 2 to 12 hours of initiating the incubation.
9 . The method of claim 2 , wherein the one or more stimulatory agents comprise (i) a first stimulatory agent that is capable of delivering the stimulatory signal and (ii) a second stimulatory agent that is capable of specifically binding to a costimulatory molecule on the one or more T cells.
10 . The method of claim 9 , wherein the first stimulatory agent specifically binds CD3 and the second stimulatory agent specifically binds CD28.
11 . The method of claim 1 , wherein the first and second stimulatory agents, independently, comprise a monovalent antibody fragment.
12 . The method of claim 11 , wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).
13 . The method of claim 1 , wherein the first stimulatory agent is an anti-CD3 Fab and the second stimulatory agent is an anti-CD28 Fab.
14 . 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 5×10 6 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.
15 . The method of claim 1 , wherein the T cells are from a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.
16 . The method of claim 1 , wherein each of the first and second stimulatory agent further comprises a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).
17 . The method of claim 1 , wherein the selection agent comprises an agent selected from the group consisting of antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes, receptor ligands, and binding fragments of any of the foregoing.
18 . The method of claim 1 , wherein:
the selection marker is a T cell coreceptor; or
the selection marker is a member of a T cell antigen receptor complex.
19 . The method of claim 1 , wherein the selection marker is selected from the group consisting of CD3, CD4, CD8, CD45RA, CD27, CD28, and CCR7.
20 . The method of claim 1 , wherein the selection agent comprises a monovalent antibody fragment.
21 . The method of claim 1 , wherein the selection agent comprises a Fab.
22 . The method of claim 4 , wherein the stimulatory reagent is an oligomeric stimulatory reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the oligomeric stimulatory reagent comprises.
23 . The method of claim 1 , wherein the oligomeric stimulatory reagent comprising a plurality of streptavidin or streptavidin mutein molecules, wherein the oligomeric stimulatory reagent comprises at least 100 streptavidin or streptavidin mutein tetramers.
24 . The method of claim 23 , wherein the oligomeric stimulatory reagent is soluble and is not bound to a solid support.
25 . The method of claim 23 , wherein the streptavidin or streptavidin mutein molecules reversibly bind to biotin, a biotin analog, or a streptavidin-binding peptide.
26 . The method of claim 23 , wherein:
the streptavidin mutein comprises the amino acid sequence Ile 44 -Gly 45 -Ala 46 -Arg 47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1; or
the streptavidin mutein comprises the amino acid sequence Val 44 -Thr 45 -Ala 46 -Arg 47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1.
27 . The method of claim 23 , wherein the streptavidin mutein comprises the amino acid sequence set forth in any of SEQ ID NOS: 3-6 and 104-105.
28 . The method of claim 23 , wherein the oligomeric stimulatory reagent comprises between 1,000 and 10,000 streptavidin or streptavidin mutein tetramers.
29 . The method of claim 23 , wherein the oligomeric stimulatory reagent is added to the stationary phase at a concentration of between about 1 μg/1 million cells to about 2 μg/1 million cells.
30 . The method of claim 1 , wherein said competition agent or free binding agent facilitates detachment of the one or more T cells from the stationary phase.
31 . The method of claim 1 , wherein said competition agent or free binding agent comprises biotin, a biotin analog, or a streptavidin-binding peptide.
32 . The method of claim 1 , further comprising introducing a recombinant nucleic acid molecule into T cells of the composition comprising stimulated T cells, wherein the nucleic acid molecule encodes a recombinant protein, thereby producing a composition comprising engineered T cells.
33 . The method of claim 32 , wherein the recombinant protein is a chimeric antigen receptor.
34 . The method of claim 32 , wherein the introduction of the recombinant nucleic acid is achieved by transduction with a viral particle.
35 . The method of claim 32 , further comprising cultivating the composition comprising engineered cells under conditions for viral integration.
36 . The method of claim 32 , further comprising cultivating the composition comprising engineered cells under conditions to expand the T cells.
37 . The method of claim 32 , further comprising harvesting the engineered T cells, thereby producing an output population of engineered T cells.
38 . The method of claim 37 , further comprising formulating the harvested cells for cryopreservation or administration to a subject.
39 . The method of claim 1 , wherein the stationary phase has a binding capacity of between about 75 million and about 125 million T cells per mL of stationary phase.