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JP-7856563-B2 - Method for producing cytotoxic effector memory T cells for cancer T cell therapy

JP7856563B2JP 7856563 B2JP7856563 B2JP 7856563B2JP-7856563-B2

Inventors

  • コンドゥリ バナジャ
  • デッカー ウィリアム ケイ．
  • ハルパート マシュー エム．
  • ヘッジ ミーナクシ ジー．
  • アーメド ナビル エム．
  • ジョセフ スジス ケイ．

Assignees

  • ベイラー カレッジ オブ メディスン

Dates

Publication Date
20260511
Application Date
20201106
Priority Date
20191106

Claims (20)

  1. An in vitro or ex vivo method for providing a population of CD8 + CD161 + T cells in which granzyme and perforin expression is upregulated, (a ) Prepare a sample that has already been collected containing CD8 + CD161 + T cells, (b) The CD8 + CD161 + T cells were cultured in the presence of IL-7, IL-15, IL-21, CD3-conjugated antibody, CD28-conjugated antibody, and Clec2d. The method comprising providing a population of CD8 + CD161 + T cells in which granzyme and perforin expression is upregulated compared to CD8+CD161+ T cells not cultured in the presence of IL-7, IL-15, IL-21, CD3-conjugated antibody, CD28-conjugated antibody, and Clec2d.
  2. (c) The method according to claim 1, further comprising culturing the CD8 + CD161 + T cells in the presence of IL-7, IL-15, and IL-21 without antibody stimulation.
  3. The method according to claim 2, wherein the culture in step (c) does not include a CD3-conjugated antibody, a CD28-conjugated antibody, and Clec2d.
  4. The method according to claim 3, wherein the culture in step (b) lasts for 12 to 72 hours, 24 to 58 hours, or 24 to 36 hours.
  5. The method according to claim 3, wherein the culture in step (c) lasts for at least 12 hours.
  6. The method according to claim 1, wherein IL-7 is present at 5 to 20 ng/ml, IL-15 is present at 2.5 to 10 ng/ml, and/or IL-21 is present at 20 to 40 ng/ml.
  7. The method according to claim 6, wherein IL-7 is present at 10 ng/ml, IL-15 is present at 5 ng/ml, and/or IL-21 is present at 30 ng/ml.
  8. The method according to claim 1, further comprising purifying or enriching T cells for the presence of CD8 + CD161 + cells in the sample prior to step (b).
  9. The method according to claim 1, further comprising, after step (b), purifying or concentrating T cells for the presence of CD8 + CD161 + cells in the sample.
  10. The method according to claim 8 or 9, wherein enriching the T cells in the sample includes fluorescent cell sorting, magnetic bead separation, or paramagnetic bead separation.
  11. The method according to any one of claims 1 to 10, wherein the culture is performed in a serum-containing culture medium.
  12. The method according to any one of claims 1 to 10, wherein the culture is performed in a serum-free medium.
  13. The method according to claim 1, wherein the cell sample is derived from the target.
  14. The method according to claim 13, wherein the sample is obtained by apheresis.
  15. The method according to claim 1, wherein the sample is a frozen and stored sample.
  16. The method according to claim 1, wherein the sample is derived from umbilical cord blood.
  17. The method according to claim 13 , wherein the sample is a peripheral blood sample from the subject.
  18. The method according to claim 1, wherein the sample is obtained by apheresis.
  19. The method according to claim 1, wherein the sample is obtained by venipuncture.
  20. The method according to any one of claims 1 to 19, further comprising introducing a nucleic acid encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) into the CD8 + CD161 + T cells in the sample.

Description

Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62/931,670, filed on November 6, 2019, the entirety of which is incorporated herein by reference. Disclaimer regarding federally funded research: This invention was made possible with government support under grant number AI127387, awarded by the National Institutes of Health. The U.S. government reserves certain rights in this invention. 1. Fields of study This disclosure generally relates to the fields of medicine, immunology, cell biology, and molecular biology. In certain aspects, the field of study of this disclosure relates to immunotherapy. More specifically, this disclosure relates to the generation of improved chimeric antigen receptor (CAR) T cells and therapeutic methods using such cells. 2. Description of Related Technologies Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive tumor with a five-year survival rate of less than 9% despite aggressive surgery, radiation, and high-dose chemotherapy (Ansari et al., 2015). In recent years, adoptive chimeric antigen receptor (CAR) T-cell therapy has shown great potential as a treatment for cancer, specifically selected CD19 + malignancies (Maude et al., 2018, Neelapu et al., 2017). CAR constructs consist of a single-chain variable region fragment (scFv) targeting cell surface tumor antigens, a transmembrane domain, a hinge region, and an intracellular signaling domain of CD3ζ typically fused to either a 4-1BB or CD28 costimulatory molecule (van der Stegen et al., 2015). In a pilot Phase I clinical trial, adoptive cell therapy using autologous mesothelin-specific CAR-T cells demonstrated safe and mild efficacy in a small number of patients with chemotherapy-refractory metastatic human PDAC (Beatty et al., 2018). However, CAR T-cell therapy for pancreatic tumors remains underdeveloped. In fact, at present, CAR-based therapies have not demonstrated any significant efficacy in solid tumors. A key feature of cell-mediated immunity against viral infection is the establishment of a population of long-lived memory T cells that provide resilient immunity to subsequent challenges through accelerated growth and cytotoxic dynamics (Seaman et al., 2004). Several groups have previously identified interesting subsets of such memory T cells, which can be identified by the expression of the innate cytotoxic receptor NK1.1 in mice or CD161 in humans (Martin et al., 2009; Turtle et al., 2009; Northfield et al., 2008; Takahashi et al., 2006; Assarsson et al., 2000; Billerbeck et al., 2010; Fergusson et al., 2011; Fergusson et al., 2016; Fergusson et al., 2014). In contrast to TCR invariant or CD8αα + CD161 + cells, polyclonal αβ cell populations exhibit stem cell-like capabilities for self-renewal and differentiation, a distinct transcriptional profile with significant upregulation of genes from the granzyme superfamily (Fergusson et al., 2011; Fergusson et al., 2014), characteristic antiviral specificity (Fergusson et al., 2008; Billerbeck et al., 2010; Havenith et al., 2012; Neelapu et al., 2005), and tissue homing properties (Billerbeck et al., 2010). Typically, CD161 is known as the innate NK cell receptor, but it can also be expressed on CD4, CD8, and NKT cells (Fergusson et al., 2016). Although found in circulation, CD8 + CD161 + cells contribute to histological pathogenesis during chronic viral infections and autoimmune states due to their tissue-resident characteristics and/or tendency to leak extravasation (Assarsson et al., 2000, Billerbeck et al., 2010, Annibali et al., 2011). Furthermore, high levels of CD161 expression in tumor-resident immune infiltration are associated with substantially improved clinical outcomes and survival in NSCLC (Braud et al., 2018). In a first embodiment, an in vitro or ex vivo method is provided, comprising (a) obtaining a cell sample comprising CD161 + T cells, and (b) culturing the T cells in the presence of IL-7, IL-15, and IL-21 to provide a population of T cells in which the number of CD161 + cells is increased compared to non-CD161 + cells. In a particular embodiment, the T cells comprise CD8 + CD161 + T cells. In a further embodiment, the T cells comprise CD4 + CD161 + T cells. IL-7 may be present at approximately 5–20 ng/ml, IL-15 at approximately 2.5–10 ng/ml, and/or IL-21 at approximately 20–40 ng/ml, for example, 10 ng/ml of IL-7, 5 ng/ml of IL-15, and/or 30 ng/ml of IL-21. The method may further include purifying or concentrating T cells for the presence of CD8 + CD161 + cells in the sample prior to step (b). The method may further include purifying or concentrating T cells for the presence of CD8 + CD161 + cells in the sample after step (b). Concentrating T cells in the sample may include fluorescent cell sorting, magnetic or paramagnetic bead separation. Culturing may be continued for up to 7, 14, 21, 28, 35, or 42 days. In some embodiments, cells are further cultured in a medium containing a CD3 and/or CD28