Methods for culturing cells and kits and apparatus for same
Inventors
Germeroth, Lothar • Stemberger, Christian • GRÄF, Patricia
Assignees
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Publication Number
US-11913024-B2
Publication Date
2024-02-27
Expiration Date
Abstract
Provided herein are methods for culturing cells, including stimulating or expanding (proliferating), a plurality of cells in a composition of cells such as a population of lymphocytes. In some aspects, provided methods and reagents for the culturing, such as stimulation or expansion (proliferation), of cell populations involve binding of agents to a molecule on the surface of the cells, thereby providing one or more signals to the cells. In some cases, the reagents are multimerization reagents and the one or more agents are multimerized by reversibly binding to the reagent. In some aspects, the multimerized agent can provide for expansion or proliferation or other stimulation of a population of cells, and then such stimulatory agents can be removed by disruption of the reversible bond. Also provided are compositions, apparatus and methods of use thereof.
Core Innovation
The invention provides a method for stimulating target T cells by reversibly immobilizing at least a plurality of target T cells on a chromatography matrix present in a chromatography column. A selection agent comprising an antibody or antibody fragment specifically binds to a selection marker expressed by the target T cells, and this specific binding effects reversible immobilization of the target T cells on the chromatography matrix.
After reversible immobilization, a soluble stimulatory reagent is added to the chromatography matrix. The stimulatory reagent comprises a first stimulatory agent and a second stimulatory agent, together with an oligomer or polymer of at least 50 crosslinked tetramers of a streptavidin mutein, wherein the first stimulatory agent comprises an anti-CD3 antibody or antibody fragment and a first streptavidin-binding peptide reversibly bound to the oligomer or polymer, and the second stimulatory agent comprises an anti-CD28 antibody or antibody fragment and a second streptavidin-binding peptide reversibly bound to the oligomer or polymer.
Incubation of the immobilized target T cells in the chromatography column in the presence of the stimulatory reagent effects stimulation of the T cells by inducing a primary activation signal via specific binding of the anti-CD3 antibody or antibody fragment to CD3 expressed on the T cells and by inducing a costimulatory signal via specific binding of the anti-CD28 antibody or antibody fragment to CD28 expressed on the T cells. The content also describes producing or enriching long-lived memory phenotypes, including central memory Tcm and stem central memory Tscm, with marker profiles including CD45RA/CD45RO, CCR7, CD62L, CD27, CD28, CD95, CD122, and KLRG1.
The document content further frames downstream use in genetically engineering cultured cells, including CAR/TCR engineering and multi-targeting, using vector delivery options, and indicates that engineered cells can be produced from or during the reversible stimulation/selection platform.
Claims Coverage
The consolidated content identifies one independent claim centered on a chromatography-column method with reversible immobilization and soluble dual-signal stimulation using a streptavidin mutein oligomer/polymer system. Multiple inventive features are present, covering reversible immobilization, soluble anti-CD3 and anti-CD28 stimulation, and incubation to induce primary activation and costimulatory signals.
Reversible T-cell immobilization on chromatography matrix via selection agent
Adding a composition comprising target T cells to a chromatography matrix present in a chromatography column, wherein a selection agent is immobilized on the chromatography matrix and comprises an antibody or antibody fragment specifically binding to a selection marker expressed by the target T cells, and wherein specific binding effects reversible immobilization of at least a plurality of the target T cells on the chromatography matrix.
Soluble dual stimulatory reagent with anti-CD3 and anti-CD28 bound reversibly to streptavidin-mutein oligomer or polymer
Adding a stimulatory reagent to the chromatography matrix in soluble form, wherein the stimulatory reagent comprises a first stimulatory agent and a second stimulatory agent, and an oligomer or polymer of at least 50 crosslinked tetramers of a streptavidin mutein, wherein the first stimulatory agent comprises an anti-CD3 antibody or antibody fragment and a first streptavidin-binding peptide reversibly bound to the oligomer or polymer, and wherein the second stimulatory agent comprises an anti-CD28 antibody or antibody fragment and a second streptavidin-binding peptide reversibly bound to the oligomer or polymer.
Incubation to induce primary activation and costimulatory signals in chromatography column
Incubating the at least a plurality of immobilized target T cells in the presence of the stimulatory reagent in the chromatography column, wherein the incubation effects stimulation of T cells and is carried out under conditions whereby the anti-CD3 antibody or antibody fragment specifically binds to CD3 expressed on the surface of the T cells to induce a primary activation signal, and the anti-CD28 antibody or antibody fragment specifically binds to CD28 expressed on the surface of the T cells to induce a costimulatory signal.
The claim coverage centers on a chromatography-column method that first reversibly immobilizes target T cells via an immobilized selection agent, then stimulates the immobilized cells using a soluble dual-signal reagent in which anti-CD3 and anti-CD28 activities are reversibly associated through streptavidin mutein oligomer or polymer and streptavidin-binding peptides to effect primary activation and costimulatory signaling.
Stated Advantages
Improves T cell expansion versus conventional anti-CD3/anti-CD28 beads.
Supports higher CD127+ long-lived memory T content after expansion (phenotype comparison described in the partial content).
Provides CD8-focused yield/phenotype effects when using αCD8 Fab addition and titration of reagent ratios (described in the partial content).
Enables selective antigen-specific expansion of CD3+ T_CM using peptide:MHC-I complexes plus αCD28, with large fold increases (described in the partial content).
Provides reversibly terminable intracellular signaling/verification, including calcium mobilization upon multimerized OKT3 Fab that is reversibly terminated by D-biotin (described in the partial content).
Enables immobilized T cell expansion within chromatography columns with reversible selection agents and multimerized CD3/CD28 stimulatory reagents, followed by elution/disruption with D-biotin (described in the partial content).
Enables stimulation and enrichment toward long-lived memory phenotypes, including central memory Tcm and stem central memory Tscm.
Provides reduced attrition, self-renewal, reduced cytotoxicity, and low TREC and low Ki-67.
Promotes a specific cytokine production pattern.
Documented Applications
Expanding human CD4+ and CD8+ responder T cells in a reversible, multimerized CD3/CD28 format, with kinetics comparisons for days 3–6 and with/without medium exchange (described in the partial content).
Phenotype analysis after expansion, including comparison of long-lived memory T content characterized by CD127+ (described in the partial content).
CD8-focused expansion/phenotyping using αCD8 Fab addition and titration of reagent ratios (described in the partial content).
Selective antigen-specific expansion of CD3+ T_CM using peptide:MHC-I complexes plus αCD28, including CMV and adenovirus epitopes (described in the partial content).
Functional verification using Jurkat cells with multimerized OKT3 Fab calcium mobilization that is reversibly terminated by D-biotin, and reversible staining/isolation (described in the partial content).
On-column culture/immobilized T cell expansion within chromatography columns using immobilized T cell expansion and reversible selection agents with multimerized CD3/CD28 stimulatory reagents, followed by elution/disruption with D-biotin (described in the partial content).
Mechanistic support in a CD19-CAR Jurkat system showing activation of intracellular signaling via reversible streptavidin/adapter binding (described in the partial content).
Stimulating target T cells using a chromatography column workflow that includes reversible immobilization and incubation with a soluble dual stimulatory reagent comprising anti-CD3 and anti-CD28 stimulatory agents linked via reversibly bound streptavidin mutein oligomer/polymer.
Genetically engineering cultured cells, including CAR/TCR engineering and multi-targeting, using vector delivery options, with engineered cells produced from or during the reversible stimulation/selection platform.
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