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EP-4739769-A1 - COMPOSITIONS AND METHODS FOR CULTURING T CELLS

EP4739769A1EP 4739769 A1EP4739769 A1EP 4739769A1EP-4739769-A1

Abstract

Cell culture medium for expanding a cell population (e.g., T cell population), cell culture compositions, and methods for culturing a cell population (e.g., T cell population) are provided. The medium can comprise a basal growth medium, a serum or serum replacement, and one or more cytokines and/or cytokine inhibitors (e.g., IL-2, IL-4, TGFβ, and/or an anti-IFNγ antibody). The method can comprising contacting the T cell population with the medium to expand the T cell population. The T cell population can comprise CD4 + and/or CD8 + primary human T cells or tumor infiltrating lymphocytes (TILs), which can be transformed to express a chimeric antigen receptor (CAR) or T cell receptor (TCR) and/or mutated by gene editing. The expanded T cell population using the medium and/or method provided herein can secrete IL-9, have increased viability, persistence, downregulation of exhaustion, mitogenesis, activation response, early memory T cell phenotype, or tumor control, and/or have an increased ratio of CD4 + /CD8 + T cells, relative to a control T cell population.

Inventors

  • CARBAJAL, Kevin
  • EKAMBARAM, Prasana
  • ANDERSON, WARREN
  • PECORARO, Adam
  • KEEVER, Mary
  • JAIN, Sumiti
  • SCHROFF, MATTHIAS
  • KRAMER, LeAnne
  • RICHTER, Maximilian

Assignees

  • Inceptor Bio, LLC

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. 1. A method of culturing a T cell population, the method comprising contacting the T cell population with an expansion medium, the expansion medium comprising a basal growth medium, a serum or serum replacement, and one or more cytokines and/or cytokine inhibitors, such that the T cell population is expanded.
  2. 2. The method of claim 1, wherein the one or more cytokines and/or cytokine inhibitors comprise IL-2, IL-4, TGFP, and/or an anti-IFNy antibody.
  3. 3. The method of claim 2, wherein said expansion medium comprise IL-2 at a concentration of 10 lU/ml or more; IL-4 at a concentration of 50 lU/ml or more; TGFP at a concentration of 3 lU/ml or more; and/or the anti-IFNy antibody at a concentration of 0-20 pg/ml.
  4. 4. The method of claim 2 or 3, wherein said expansion medium comprises TGFP at a concentration of 4.5 lU/ml or more.
  5. 5. The method of any one of claims 1-4, wherein said expansion medium does not comprise the anti-IFNy antibody or IL-4.
  6. 6. The method of any one of claims 1-5, wherein the T cell population is not contacted with a medium containing IL-2 and no other cytokines or cytokine inhibitors.
  7. 7. The method of any one of claims 1-6, wherein the basal growth medium comprises a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and/or IMMUNOCULT™-XF T Cell Expansion Medium.
  8. 8. The method of any one of claims 1-7, wherein the serum or serum replacement comprises fetal bovine serum (FBS), human AB serum, CTS™ Immune Cell Serum Replacement, and/or PHYSIOLOGIX™ Xeno-Free Serum Replacement.
  9. 9. The method of any one of claims 1-8, wherein the T cell population comprises one or more primary human T cells.
  10. 10. The method of any one of claims 1-9, wherein the T cell population consists essentially of one or more CD4 + and/or CD8 + T cells.
  11. 11. The method of any one of claims 1-10, wherein the T cell population comprises one or more tumor infiltrating lymphocytes (TILs).
  12. 12. The method of any one of claims 1-11, wherein the expansion medium is replaced with fresh expansion medium every 2-3 days and/or the T cell population is cultured in the expansion medium for up to about 14 days.
  13. 13. The method of any one of claims 1-12, comprising culturing the T cell population in the expansion medium for at least 3 days optionally followed by culturing the T cell population in a control medium, or comprising culturing the T cell population in the expansion medium for at least 7 days optionally after culturing the T cell population in a control medium.
  14. 14. The method of any one of claims 1-13, the method further comprising, after culturing the T cell population in the expansion medium, freezing the T cell population and/or administering the T cell population to a subject.
  15. 15. The method of any one of claims 1-14, wherein the T cell population is cultured in a system comprising a gas permeable membrane culture surface.
  16. 16. The method of any one of claims 1-15, the method further comprising, prior to contacting the T cell population with the expansion medium: (a) thawing a plurality of cells comprising the T cell population from a frozen stock; (b) selecting from the plurality of cells one or more CD4 + and/or CD8 + T cells as the T cell population; (c) contacting the T cell population with an activating reagent such that the T cell population is activated; (d) introducing a polynucleotide encoding a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR) into the T cell population such that one or more cells of the T cell population expresses the CAR or the TCR; and/or (e) contacting the T cell population with one or more gene editing reagents such that a mutation is introduced at a target site in a genome of one or more cells of the T cell population and level or activity of a gene of interest is altered in the one or more cells.
  17. 17. The method of claim 16, wherein: said step of selecting one or more CD4 + and/or CD8 + T cells is performed within one day after the step of thawing a plurality of cells; said step of contacting the T cell population with an activating reagent is performed within one day after the step of selecting one or more CD4 + and/or CD8 + T cells; said step of introducing a polynucleotide encoding a CAR and/or a TCR into the T cell population is performed about 1-2 days after the step of contacting the T cell population with an activating reagent; said step of contacting the T cell population with one or more gene editing reagents is performed about 3 days after the step of contacting the T cell population with an activating reagent and/or about 1-2 days after the step of introducing a polynucleotide encoding a CAR and/or a TCR into the T cell population; and/or said step of contacting the T cell population with expansion medium is performed within one day after the step of introducing a polynucleotide encoding a CAR and/or a TCR into the T cell population or the step of contacting the T cell population with one or more gene editing reagents.
  18. 18. The method of claim 16 or 17, wherein said step of selecting one or more CD4 + and/or CD8 + T cells comprises contacting the plurality of cells with magnetic beads coupled with an anti- CD4 + antibody and an anti-CD8 + antibody, and selecting CD4 + and/or CD8 + T cells that are bound to the magnetic beads.
  19. 19. The method of any one of claims 16-18, wherein one or more cells of the T cell population are CD3 + and CD28 + , and the activating reagent binds and crosslinks CD3 and CD28 on surface of the one or more cells of the T cell population.
  20. 20. The method of any one of claims 16-19, wherein introducing the polynucleotide encoding a CAR and/or a TCR into the T cell population comprises introducing a viral vector comprising the polynucleotide into the T cell population.

Description

COMPOSITIONS AND METHODS FOR CULTURING T CELLS RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/512,006, filed on July 5, 2023, and U.S. Provisional Application No. 63/643,819, filed on May 7, 2024. The entire contents of each of the foregoing applications are incorporated herein by reference. FIELD OF THE DISCLOSURE The disclosure relates to culturing and expansion of T cells, e.g., primary human T cells. BACKGROUND OF THE DISCLOSURE Cell therapies, such as T cell receptor (TCR) therapy, adoptive cell therapy (ACT), chimeric antigen receptor (CAR)-T cell therapy, and tumor-infiltrating lymphocytes (TIL) therapy, is effective for treatment of malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion or viability, antigen loss, and limited persistence of infused T cells. Limited persistence of T cells can be due to programmed cell death and exhaustion. Methods for efficiently culturing and expanding cells (e.g., T cells) and culturing and expanding cells (e.g., T cells) that persist in cancer therapy are therefore needed. BRIEF SUMMARY OF THE DISCLOSURE A cell culture medium for expanding a cell population (e.g., a T cell population), a cell composition comprising the cell culture medium and a population of cells cultured therein, and a method for culturing a cell population (e.g., a T cell population) is provided. The medium can comprise a basal growth medium, a serum or serum replacement, and one or more cytokines and/or cytokine inhibitors (e.g., IL-2, IL-4, TGFP, and/or an anti-IFNy antibody). The method can comprise contacting the T cell population with the medium to expand the T cell population. The T cell population can comprise CD4+ and/or CD8+ primary human T cells, or tumor infiltrating lymphocytes (TILs), which can be transformed to express any target-specific receptors, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR), and/or mutated by gene editing. The expanded T cell population using the medium and/or method provided herein can secrete IL-9; express CCR4, CCR7, CD62L, and/or CD45RA; have increased viability, persistence, downregulation of exhaustion, and/or tumor control; have increased mitogenesis in response to a target antigen; have an early memory T cell phenotype or prolonged survival; have less differentiation into the effector phenotype (i.e., have a younger phenotype) or less T cell exhaustion; have enhanced proliferation or activation regardless of suppression by TGFP; have decreased potentiation of the TGFP signaling pathway in response to stimuli; do not require IL-2 for proliferation; and/or have an increased ratio of CD4+/CD8+ T cells, relative to a control T cell population (e.g., cultured using conventional T medium). In one aspect, the present disclosure provides a method for culturing a T cell population. The method includes contacting the T cell population with expansion medium containing a basal growth medium, a serum or serum replacement, and one or more cytokines and/or cytokine inhibitors, such that the T cell population is expanded. In some embodiments, the one or more cytokines and/or cytokine inhibitors in the expansion medium comprise IL-2, IL-4, TGFP, and/or an anti-IFNy antibody. In some embodiments, the expansion medium comprise IL-2 at a concentration of 10 lU/ml or more (e.g., 10-400 lU/ml); IL-4 at a concentration of 50 lU/ml or more (e.g., 250-500 lU/ml); TGFP at a concentration of 3 lU/ml or more (e.g., 20-150 lU/ml); and/or the anti-IFNy antibody at a concentration of 0 pg/ml or more (e.g., 0-20 pg/ml). In specific embodiments, the expansion medium comprises TGFP at a concentration of 4.5 lU/ml or more. In some embodiments, the expansion medium does not comprise the anti-IFNy antibody or IL-4. In some embodiments, the T cell population is not contacted with a medium or medium containing IL-2 and no other cytokines or cytokine inhibitors. In some embodiments, the basal growth medium comprises a Roswell Park Memorial Institute (RPMI) 1640 medium, X-VIVO™-15 medium, CTS™ OPTMIZER™ Serum Free Medium, CTS™ OPTMIZER™ Pro Serum Free Medium, and/or IMMLJNOCLrLT™-XF T Cell Expansion Medium. In some embodiments, the serum or serum replacement comprises fetal bovine serum (FBS), human AB serum, CTS™ Immune Cell Serum Replacement, and/or PHYSIOLOGIX™ Xeno-Free Serum Replacement. In some embodiments, the T cell population comprise one or more primary T cells, such as one or more primary human T cells. In some embodiments, the T cell population consists essentially of one or more CD4+ and/or CD8+ T cells. In some embodiments, the T cell population comprises one or more tumor infiltrating lymphocytes (TILs). In some embodiments, the expansion medium is replaced with fresh expansion medium every 2-3 days and/or the T cell population is cultured in the expansion medium for up to about 14 days. In some embodiments, the method includes culturing the T cell