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US 2021/0171909 A1Published application
Methods of making chimeric antigen receptor?expressing cells
AssigneeNovartis AG
InventorTatiana Golovina
Life of this patent
Estimating the term…
Abstract
The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that express a chimeric antigen receptor (CAR), and compositions generated by such methods.
Claims61
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- (i) contacting a population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, and
- (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, wherein:
- the population of cells at the beginning of step (i) has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) of the following properties:
- (1) the population of cells at the beginning of step (i) does not expand or expands for no more than 5, 6, 7, 8, or 9-fold over 8-11 days using the Bead CART cell manufacturing process described in Example 1,
- (2) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells in the population of cells at the beginning of step (i) is lower than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60%, e.g., lower than 10%,
- (3) the percentage of naïve T cells and/or Tscm among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells in the population of cells at the beginning of step (i) is lower than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, or 90% lower than the corresponding value in a reference population of cells,
- (4) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells in the population of cells at the beginning of step (i) is higher than 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95%, e.g., higher than 50%,
- (5) the percentage of Teff cells and/or Tem cells among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells in the population of cells at the beginning of step (i) is higher than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900% higher than the corresponding value in a reference population of cells,
- (6) the percentage of CD28+CD4+ T cells among CD4+ T cells in the population of cells at the beginning of step (i) is no more than 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, e.g., no more than 50%,
- (7) the percentage of CD28+CD8+ T cells among CD8+ T cells in the population of cells at the beginning of step (i) is no more than 30, 35, 40, 45, 50, 55, 60, 65, or 70%, e.g., no more than 50%,
- (8) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells in the population of cells at the beginning of step (i) is lower than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CART cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, or 90% lower than the corresponding value in a reference population of cells,
- (9) the ratio of CD4+ T cells to CD8+ T cells in the population of cells at the beginning of step (i) is lower than 0.5, 0.8, 1, 1.2, or 1.5,
- (10) the ratio of CD4+ T cells to CD8+ T cells in the population of cells at the beginning of step (i) is lower than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 99% lower than the corresponding value in a reference population of cells,
- (11) the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells in the population of cells at the beginning of step (i) is more than 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95%,
- (12) the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells in the population of cells at the beginning of step (i) is more than 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 95%,
- (13) the percentage of senescent T cells (e.g., CD28−CD27−CD57+ senescent T cells) among T cells, the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells, or the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells in the population of cells at the beginning of step (i) is higher than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 5000, or 9500% higher than the corresponding value in a reference population of cells,
- (14) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells in the population of cells at the beginning of step (i) is higher than 10, 20, 30, 40, 50, 60, 70, 80 or 90%, and
- (15) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells in the population of cells at the beginning of step (i) is higher than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 5000 or 9000% higher than the corresponding value in a reference population of cells.
16 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- (a) providing apheresis, e.g., leukapheresis, material, optionally wherein the apheresis, e.g., leukapheresis, material is cryopreserved after being isolated from a subject, and thawed prior to step (b),
- (b) isolating a population of cells (e.g., T cells) from the apheresis, e.g., leukapheresis, material using negative selection, e.g., by reducing monocytes (e.g., CD14+ cells), B cells (e.g., CD19+ cells), and/or NK cells (e.g., CD56+ cells), from the apheresis, e.g., leukapheresis, material, e.g., using CliniMACS,
- (c) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (d) contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, e.g., contacting (e.g., binding) the population of cells (e.g., T cells) with T Cell TransAct™,
- (e) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (f) expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days, to produce a population of expanded cells, optionally wherein:
- the population of cells at the beginning of step (i) has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) of the following properties:
- (1) the population of cells at the beginning of step (i) does not expand or expands for no more than 5, 6, 7, 8, or 9-fold over 8-11 days using the Bead CART cell manufacturing process described in Example 1,
- (2) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells in the population of cells at the beginning of step (i) is lower than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60%, e.g., lower than 10%,
- (3) the percentage of naïve T cells and/or Tscm among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells in the population of cells at the beginning of step (i) is lower than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, or 90% lower than the corresponding value in a reference population of cells,
- (4) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells in the population of cells at the beginning of step (i) is higher than 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95%, e.g., higher than 50%,
- (5) the percentage of Teff cells and/or Tem cells among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells in the population of cells at the beginning of step (i) is higher than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 or 900% higher than the corresponding value in a reference population of cells,
- (6) the percentage of CD28+CD4+ T cells among CD4+ T cells in the population of cells at the beginning of step (i) is no more than 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, e.g., no more than 50%,
- (7) the percentage of CD28+CD8+ T cells among CD8+ T cells in the population of cells at the beginning of step (i) is no more than 30, 35, 40, 45, 50, 55, 60, 65, or 70%, e.g., no more than 50%,
- (8) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells in the population of cells at the beginning of step (i) is lower than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CART cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, or 90% lower than the corresponding value in a reference population of cells,
- (9) the ratio of CD4+ T cells to CD8+ T cells in the population of cells at the beginning of step (i) is lower than 0.5, 0.8, 1, 1.2, or 1.5,
- (10) the ratio of CD4+ T cells to CD8+ T cells in the population of cells at the beginning of step (i) is lower than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 99% lower than the corresponding value in a reference population of cells,
- (11) the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells in the population of cells at the beginning of step (i) is more than 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95%,
- (12) the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells in the population of cells at the beginning of step (i) is more than 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 95%,
- (13) the percentage of senescent T cells (e.g., CD28−CD27−CD57+ senescent T cells) among T cells, the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells, or the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells in the population of cells at the beginning of step (i) is higher than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 60, 70, 80, 90 or 95% higher than the corresponding value in a reference population of cells,
- (14) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells in the population of cells at the beginning of step (i) is higher than 10, 20, 30, 40, 50, 60, 70, 80 or 90%, and
- (15) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells in the population of cells at the beginning of step (i) is higher than the corresponding value in a reference population of cells (e.g., a population of cells from a healthy donor, or a population of cells that expands more than 10, 15, or 20-fold over 8-11 days using the Bead CAR T cell manufacturing process described in Example 1), e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 5000 or 9000% higher than the corresponding value in a reference population of cells.
20 more elements
A method of evaluating or predicting suitability of a population of cells (e.g., T cells) for chimeric antigen receptor (CAR) manufacturing, the method comprising:
- acquiring a value for one or more (e.g., 2, 3, 4, 5, or all) of the following from the population of cells (e.g., T cells):
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells,
- (2) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells,
- (3) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells,
- (4) the ratio of CD4+ T cells to CD8+ T cells,
- (5) the percentage of senescent T cells (e.g., CD28−CD27−CD57+ senescent T cells) among T cells, the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells, or the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells, and
- (6) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells, wherein:
- (a) a decrease in the value of one, two, or all of (1), (3), and (4) as compared to a reference value, e.g., a healthy donor reference value, is indicative or predicative of decreased suitability of the population of cells (e.g., T cells) for CAR manufacturing,
- (b) an increase in the value of one, two, or all of (1), (3), and (4) as compared to a reference value, e.g., a healthy donor reference value, is indicative or predicative of increased suitability of the population of cells (e.g., T cells) for CAR manufacturing,
- (c) an increase in the value of one, two, or all of (2), (5), and (6) as compared to a reference value, e.g., a healthy donor reference value, is indicative or predicative of decreased suitability of the population of cells (e.g., T cells) for CAR manufacturing, or
- (d) a decrease in the value of one, two, or all of (2), (5), and (6) as compared to a reference value, e.g., a healthy donor reference value, is indicative or predicative of increased suitability of the population of cells (e.g., T cells) for CAR manufacturing,
- thereby evaluating or predicting suitability of the population of cells (e.g., T cells) for CAR manufacturing.
9 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- responsive to a decreased value for one, two, or all of the following in the population of cells (e.g., T cells):
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells,
- (2) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells, and
- (3) the ratio of CD4+ T cells to CD8+ T cells,
- and/or responsive to an increased value of one, two, or all of the following in the population of cells (e.g., T cells):
- (4) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells,
- (5) the percentage of senescent T cells (e.g., CD28−CD27−CD57+ senescent T cells) among T cells, the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells, or the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells, and
- (6) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells,
- as compared to a reference value, e.g., a healthy donor reference value, performing:
- (a) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (b) optionally, contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, optionally wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix, optionally wherein the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™,
- (c) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (d) optionally, expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days in vitro, to produce a population of expanded cells, optionally wherein:
- performing the bead-free stimulation and cytokine (BFSC) process described in Example 1.
12 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- responsive to:
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells in the population of cells (e.g., T cells) being lower than 8, 9, 10, 11, 12, 15, or 20%, e.g., 10%,
- (2) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells in the population of cells (e.g., T cells) being higher than 40, 45, 50, 55, or 60%, e.g. 50%, and/or
- (3) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells in the population of cells (e.g., T cells) being lower than 40, 45, 50, 55, or 60%, e.g. 50%, performing:
- (a) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (b) optionally, contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, optionally wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix, optionally wherein the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™,
- (c) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (d) optionally, expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days in vitro, to produce a population of expanded cells, optionally wherein:
- performing the bead-free stimulation and cytokine (BFSC) process described in Example 1.
7 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- responsive to:
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells in the population of cells (e.g., T cells) being higher than 8, 9, 10, 11, 12, 15, or 20%, e.g., 10%,
- (2) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells in the population of cells (e.g., T cells) being lower than 40, 45, 50, 55, or 60%, e.g. 50%, and/or
- (3) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells in the population of cells (e.g., T cells) being higher than 40, 45, 50, 55, or 60%, e.g. 50%, performing:
- (A) (a) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (b) optionally, contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, optionally wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix, optionally wherein the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™,
- (c) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (d) optionally, expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days in vitro, to produce a population of expanded cells, optionally wherein:
- performing the bead-free stimulation and cytokine (BFSC) process described in Example 1, or
- (B) the Bead CAR T cell manufacturing process described in Example 1.
8 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- acquiring a value for one or more (e.g., 2, 3, 4, 5, or all) of the following from the population of cells (e.g., T cells):
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells,
- (2) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells,
- (3) the ratio of CD4+ T cells to CD8+ T cells,
- (4) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells,
- (5) the percentage of senescent T cells (e.g., CD28−CD27−CD57+ senescent T cells) among T cells, the percentage of CD4+ senescent cells (e.g., CD28−CD27−CD57+CD4+ senescent cells) among CD4+ T cells, or the percentage of CD8+ senescent cells (e.g., CD28−CD27−CD57+CD8+ senescent cells) among CD8+ T cells, and
- (6) the percentage of exhausted T cells (e.g., PD-1+, LAG3+, or PD-1+LAG3+ T cells) among T cells, the percentage of exhausted CD4+ T cells (e.g., PD-1+CD4+, LAG3+CD4+, or PD-1+LAG3+CD4+ T cells) among CD4+ T cells, or the percentage of exhausted CD8+ T cells (e.g., PD-1+CD8+, LAG3+CD8+, or PD-1+LAG3+CD8+ T cells) among CD8+ T cells, wherein:
- responsive to a decrease in the value of one, two, or all of (1)-(3), or responsive to an increase in the value of one, two, or all of (4)-(6), as compared to a reference value, e.g., a healthy donor reference value, performing:
- (a) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (b) optionally, contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, optionally wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix, optionally wherein the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™,
- (c) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (d) optionally, expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days in vitro, to produce a population of expanded cells, optionally wherein:
- performing the bead-free stimulation and cytokine (BFSC) process described in Example 1.
11 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- acquiring a value for one or more (e.g., 2 or all) of the following from the population of cells (e.g., T cells):
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells,
- (2) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells, and
- (3) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells, wherein:
- responsive to value (1) being lower than 8, 9, 10, 11, 12, 15, or 20%, e.g., 10%;
- value (2) being higher than 40, 45, 50, 55, or 60%, e.g. 50%;
- and/or value (3) being lower than 40, 45, 50, 55, or 60%, e.g. 50%, performing:
- (a) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (b) optionally, contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, optionally wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix, optionally wherein the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™,
- (c) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (d) optionally, expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days in vitro, to produce a population of expanded cells, optionally wherein:
- performing the bead-free stimulation and cytokine (BFSC) process described in Example 1.
10 more elements
A method of making a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising:
- acquiring a value for one or more (e.g., 2 or all) of the following from the population of cells (e.g., T cells):
- (1) the percentage of naïve T cells and/or stem cell-like memory T cells (Tscm) among T cells, the percentage of CD4+ naïve T cells and/or Tscm cells among CD4+ T cells, or the percentage of CD8+ naïve T cells and/or Tscm cells among CD8+ T cells,
- (2) the percentage of effector T cells (Teff) and/or effector memory T cells (Tem) among T cells, the percentage of CD4+ Teff cells and/or Tem cells among CD4+ T cells, or the percentage of CD8+ Teff cells and/or Tem cells among CD8+ T cells, and
- (3) the percentage of CD28+ T cells among T cells, the percentage of CD28+CD4+ T cells among CD4+ T cells, or the percentage of CD28+CD8+ T cells among CD8+ T cells, wherein:
- responsive to value (1) being higher than 8, 9, 10, 11, 12, 15, or 20%, e.g., 10%;
- value (2) being lower than 40, 45, 50, 55, or 60%, e.g. 50%;
- and/or value (3) being higher than 40, 45, 50, 55, or 60%, e.g. 50%, performing:
- (A) (a) contacting the population of cells (e.g., T cells) with IL-2 and one or both of:
- IL-15 and IL-21, e.g., contacting the population of cells (e.g., T cells) with IL-2, IL-15, and IL-21,
- (b) optionally, contacting (e.g., binding) the population of cells (e.g., T cells) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule, optionally wherein the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix, optionally wherein the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™,
- (c) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and
- (d) optionally, expanding the population of cells comprising the nucleic acid molecule for at least 3, 4, 5, 6, 7, 8, 9, or 10 days in vitro, e.g., expanding the population of cells comprising the nucleic acid molecule for about 4-10 days in vitro, to produce a population of expanded cells, optionally wherein:
- performing the bead-free stimulation and cytokine (BFSC) process described in Example 1, or
- (B) the Bead CAR T cell manufacturing process described in Example 1.
11 more elements
Dependencies are inferred from the claim text.