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US-12617864-B2 - Methods of activating and proliferating exhausted CD8 T cells, CD8 T cells with enhanced activity prepared by the same, and uses thereof

US12617864B2US 12617864 B2US12617864 B2US 12617864B2US-12617864-B2

Abstract

The present invention relates to a method for activating a cell and a cell activated thereby and a use thereof, more particularly, to an in vitro method of enhancing, recovering of immune response of CD8 T cells in exhaustion and proliferating the CD8 T cells comprising the step of inducing overexpression of Klf4 protein in CD8 T cells, a cell population containing the CD8 T cells or transduced CAR-CD8 T cells whose anticancer activity is enhanced by overexpressing Klf4 protein and use thereof.

Inventors

  • Rho Hyun SEONG
  • Jin Woo NAH

Assignees

  • Medgene Therapeutics, Inc.

Dates

Publication Date
20260505
Application Date
20220412
Priority Date
20210412

Claims (20)

  1. 1 . An in vitro method of suppressing the exhaustion state of CD8 T cells comprising inducing overexpression of Klf4 protein in CD8 T cell-containing cells selected from the group consisting of a) CD8 T cells, b) a cell population comprising the CD8 T cells, and c) transduced CAR-CD8 T cells prepared by transducing the CD8 T cells with a gene encoding a chimeric antigen receptor (CAR), wherein the overexpression of the Klf4 protein is performed by transfecting the CD8 T cell-containing cells with an expression vector containing a polynucleotide encoding the Klf4 protein, or introducing an mRNA expressing the Klf4 protein into the CD8 T cell-containing cells.
  2. 2 . The method according to claim 1 , wherein the CD8 T cells are autologous cells isolated from a subject in need of treatment or heterologous cells isolated from other subject.
  3. 3 . The method according to claim 1 , wherein the expression vector is a viral vector or a non-viral vector.
  4. 4 . The method according to claim 3 , wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, or a vaccinia virus vector, Sendaivirus vector, flavivirus vector, radovirus vector, retroviral vector, herpesvirus vector, poxvirus vector or lentiviral vector.
  5. 5 . The method according to claim 3 , wherein the non-viral vector is mRNA, a DNA vector, nanoparticles, cationic polymer, exosome, extracellular vesicle or liposome.
  6. 6 . The method according to claim 5 , wherein the DNA vector is a plasmid vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.
  7. 7 . The method according to claim 5 , wherein the mRNA comprises a polynucleotide encoding a Klf4 protein, and the mRNA is used alone or in combination with a non-viral vector other than the mRNA.
  8. 8 . The method according to claim 1 , wherein the expression vector further comprises a polynucleotide encoding one or more immune-stimulating peptides.
  9. 9 . The method according to claim 8 , wherein the immune-stimulating peptide is CD28, ICOS (inducible costimulator), CTLA4 (cytotoxic T lymphocyte associated protein 4), PD1 (programmed cell death protein 1), BTLA (B and T lymphocyte associated protein), DR3 (death receptor 3), 4-1BB, CD2, CD40, CD40L, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKG2D (natural-killer group 2, member D)/DAP12 (DNAX-activating protein 12), TIM1 (T-cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1, B7-H1, GITR (glucocorticoid-induced TNFR family related protein), Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, herpesvirus entry mediator (HVEM), or the cytoplasmic domain of OX40L [ligand for CD134 (OX40), CD252], or a linkage of two or more thereof.
  10. 10 . A pharmaceutical composition for treating cancer comprising CD8 T cells whose overexpression of Klf4 protein is induced as an effective ingredient, wherein the CD8T cells whose overexpression of Klf4 protein is induced are prepared by transfecting the CD8 T cell with (a) an expression vector containing a polynucleotide encoding the Klf4 protein or (b) an mRNA expressing the Klf4 protein.
  11. 11 . The pharmaceutical composition according to claim 10 , wherein the CD8 T cells are autologous cells isolated from a subject in need of treatment or heterologous cells isolated from other subject.
  12. 12 . A transduced CD8 T cell which is transformed to overexpress the Klf4 protein, wherein the transduced CD8 T cell is prepared by transfecting the CD8 T cell with (a) an expression vector containing a polynucleotide encoding the Klf4 protein or (b) an mRNA expressing the Klf4 protein.
  13. 13 . The transduced CD8 T cell according to claim 12 , wherein the expression vector is a viral vector or a non-viral vector.
  14. 14 . The transduced CD8 T cell according to claim 13 , wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, or a Sendai virus vector, a flavivirus vector, a radovirus vector, a retroviral vector, a herpesvirus vector, a poxvirus vector or a lentiviral vector.
  15. 15 . The transduced CD8 T cell according to claim 13 , wherein the non-viral vector is mRNA, a DNA vector, nanoparticles, cationic polymer, exosome, extracellular vesicle, or a liposome.
  16. 16 . The transduced CD8 T cell according to claim 15 , wherein the DNA vector is a plasmid vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.
  17. 17 . The transduced CD8 T cell according to claim 15 , wherein the mRNA comprises a polynucleotide encoding a Klf4 protein, and the mRNA is used alone or in combination with a non-viral vector other than the mRNA.
  18. 18 . A composition comprising the transduced CD8 T cell of claim 12 .
  19. 19 . A transduced CAR-CD8 T cell prepared by transducing a CD8 T cell isolated from a subject in need thereof or a cell population containing the CD8 T cell whose overexpression of Klf4 protein is induced with a gene encoding a chimeric antigen receptor (CAR), wherein the transduced CD8 T cell is prepared by transfecting the CD8 T cell with (a) an expression vector containing a polynucleotide encoding the Klf4 protein or (b) an mRNA expressing the Klf4 protein.
  20. 20 . The transduced CAR-CD8 T cell according to claim 19 , wherein the CAR is a fusion protein comprising a single chain-based antibody mimetic, a transmembrane domain, a costimulatory domain and a cytoplasmic signaling domain.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. application Ser. No. 17/478,445, filed Sep. 17, 2021, which claims priority to KR Appl. No. 10-2021-0047002, filed Apr. 12, 2021, both of which are incorporated herein by reference in their entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The content of the electronically submitted sequence listing in ASCII text file (Name: 2486-0003US02_Sequence Listing_ST25. txt; Size: 25.4 KB; and Date of Creation: Apr. 12, 2022) filed with the application is incorporated herein by reference in its entirety. TECHNICAL FIELD The present invention relates to a method of cell proliferation and activation, a cell activated thereby and a use thereof, and more particularly, to a method of activating and proliferating CD8 T cells continuously exposed to an antigen, and CD8 T cells with enhanced anticancer activity produced by the same and uses thereof. BACKGROUND OF THE INVENTION Most CD8 T cells infiltrated the tumor tissue recognize specific antigens derived from cancer cells. The antigen (Ag)-activated CD8 T cells can eliminate cancer cells by secreting effector molecules such as granzyme B, interferon-gamma (IFN-γ), and perforin. Therefore, increasing the activity of the CD8 T cells specific for cancer antigens is regarded as one of the most efficient approaches to treat cancers. Check point inhibitors such as anti-PD-1 and anti-CTLA antibodies (Abs) work to control cancers in such a way, however, the efficiency is quite limited. It is recognized that controlling the CD8 T cell activity in cancer tissues is highly sophisticated because of the complex characteristics of cancer microenvironment. In the cancer microenvironment, antigen-specific CD8 T cells are continuously exposed to cancer antigens unless cancer is completely removed. Chronic antigen stimulation makes CD8 T cells fall into unresponsive state which is currently described as ‘exhaustion’. Exhausted CD8 T cells are not able to efficiently eliminate cancer cells because they cannot perform a normal immune response due to the weakening of active cytokine secretion and cell division functions. In particular, these exhausted CD8 T cells have a characteristic that they do not easily return to cells with normal functions even after time passes. Therefore, preventing CD8 T cells from being exhausted in the tumor tissue and reactivating the function of the exhausted cells is critical to control cancers. Recent studies have shown that CD8 T cells from chronic virus infected tissues or tumor tissues are composed of 4 subsets of cell groups displaying stages of exhaustion; These include chronic progenitor cell subset (Progenitor exhausted cells 1, Progenitor exhausted cells 2), chronic effector cell subset (intermediate exhausted cells) and end-stage exhausted cell subset (terminal exhausted cells). Among them, chronic effector cell subset has the highest effector function to recognize and remove cancer cells, but terminal exhausted cells are known to have lost a significant part of an effector function. Indeed, it is known that most of the CD8 T cells present in cancer tissues do not function properly since they are in the terminal state of exhaustion. Therefore, if a method for enhancing the generation and function of chronic effector cells from cancer-specific CD8 T cells is developed, it will be very useful to effectively control and treat cancer. With respect to anticancer treatment using CD8 T cells, U.S. Pat. No. 8,106,092 discloses a method of treating secondary cancer by inducing necrosis of cancer cells and promoting generation of cancer-specific T cells such as CD8 T cells, comprising administrating ingenol-3-angelate locally and/or intratumorally to an individual with secondary cancer, and US Patent Publication No. US20190218515A discloses a method of producing activated T cells comprising treating T cells isolated from cancer patients with a dual specific antibody against CD123/CD3, a dual-specific antibody against CD19/CD3 or a dual-specific antibody against EpCAM/CD3. On the other hand, T cell therapeutics are being developed up to the 3rd generation so far. The first-generation T cell therapeutics have low specificity for cancer cells because they are administered to patients by proliferating all T cells (bulk T cells) present in the blood or cancer tissue. Efficacy could not be expected, and the second-generation T cell therapeutics showed an improved therapeutic effect by isolating/mass-culturing only tumor antigen-specific T cells and administering them to cancer patients. The problem of this long and complicated process has been raised, and the third-generation T cell therapeutics have been developed by either 1) directly introducing a TCR gene that recognizes a specific cancer antigen into T cells, or 2) preparing a fusion protein by linking an antigen recognition site (scFv) of a monoclonal antibody that recognizes a specific antigen to a T cell