Search

EP-4735581-A2 - METHODS OF MAKING AND USING ENGINEERED IMMUNE CELLS

EP4735581A2EP 4735581 A2EP4735581 A2EP 4735581A2EP-4735581-A2

Abstract

The disclosure provides for a cell comprising an inactivating modification in the FAS and/or FASL gene; wherein the cell does not express a functional FAS and/or FASL protein. Also described is a method for making a cell, the method comprising culturing a cell of the disclosure under conditions suitable for the in vitro differentiation of the cell into a mature cell. Also provided are cells produced by the methods of the disclosure as well as a method for treating a subject comprising administering cells of the disclosure.

Inventors

  • SEET, Christopher S.
  • LI, Suwen

Assignees

  • The Regents of University of California

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A cell comprising an inactivating modification in the FAS (Fas cell surface death receptor) and/or FAS ligand (FASL) gene; wherein the cell does not express a functional FAS and/or FASL protein.
  2. 2. The cell of claim 1, wherein the FAS gene and/or FASL gene are modified using gene editing to generate inactive FAS and/or FASL genes.
  3. 3. The cell of claim 2, wherein the FAS and/or FASL gene are modified by contacting the cells with a site-specific Cas nuclease and a guide RNA.
  4. 4. The cell of any one of claims 1-3, wherein the cell further comprise a heterologous nucleic acid encoding a transgene.
  5. 5. The cell of any one of claims 1-4, wherein the transgene comprises a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR).
  6. 6. The cell of claim 5, wherein the CAR comprises an anti-CD19 CAR.
  7. 7. The cell of claim 5, wherein the TCR comprises an anti-NY-ESOl TCR.
  8. 8. The cell of any one of claims 4-7, wherein the cell expresses the transgene.
  9. 9. The cell of any one of claims 1-8, wherein the cell comprises a stem cell, a hematopoietic stem or progenitor cell (HSPC), embryonic stem cells, an induced pluripotent stem cell (iPSC), human embryonic mesodermal progenitor cells, or a pluripotent stem cell (PSC).
  10. 10. The cell of claim 9, wherein the cell comprises a pluripotent stem cell.
  11. 11. The cell of claim 9, wherein the cell comprises an embryonic stem cell or an induced pluripotent stem cell (iPSC).
  12. 12. The cell of any one of claims 1-11, wherein the cell is isolated or derived from cord blood, peripheral blood, bone marrow, peripheral blood, umbilical cord blood, placenta, adipose tissue, or umbilical cord tissue.
  13. 13. The cell of any one of claims 1-12, wherein the cell comprises a T cell.
  14. 14. The cell of claim 13, wherein the T cell comprises a peripheral blood T cell.
  15. 15. The cell of any one of claims 1-14, wherein the cell comprises a mature cell.
  16. 16. The cell of claim 15, wherein the mature cell comprises a mature T cell, mature regulatory T cell (Treg), an iNKT cell, an innate lymphoid cell, or NK cell.
  17. 17. The cell of claim 16, wherein the cell comprises a CD4+ CD8- or CD8ab+ CD4- T cell.
  18. 18. The cell of any one of claims 13-17, wherein the cell comprises an in vitro differentiated T cell, mature Treg, iNKT cell, innate lymphoid cell, or NK cell.
  19. 19. The cell of any one of claims 1-18, wherein the cell is further defined as allogeneic.
  20. 20. A method for making a cell comprising culturing the cell of any one of claims 1-19 under conditions suitable for the in vitro differentiation of the cell into a mature cell.

Description

METHODS OF MAKING AND USING ENGINEERED IMMUNE CELLS BACKGROUND [0001] This application claims priority of U.S. Provisional Patent Application No. 63/524,313, filed June 30, 2023, which is hereby incorporated by reference in its entirety. SEQUENCE LISTING [0002] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on June 28, 2024, is named UCLAP0182WO.xml, and is 69,632 bytes in size. GOVERNMENT SUPPORT CLAUSE [0003] This invention was made with government support under CA235525 awarded by the National Institutes of Health. The government has certain rights in the invention. I. Field of the Invention [0004] This invention relates to the field of medicine and molecular biology II. Background [0005] The use of stem cells or progenitor cells as a source for in vitro generation of T cells for adoptive cell therapy offers the potential for an alternative to the current approaches that rely on manipulation of mature peripheral blood T cells. The generation of T cells from pluripotent stem cells (PSC) or hematopoietic stem and progenitor cells (HSPC) offers an advantage over the use of primary mature T cells harvested from the blood because of the ability of stem cells to self-renew indefinitely either in vivo (in the case of HSPC) or in vitro (in the case of PSC), as well as the opportunity for complex gene modifications in stem cells to impart therapeutically favorable properties to their progeny T cells. There is a need in the art to provide for improved methods that increase cell yield and/or therapeutic efficacy of T cells produced from these stem cell sources. BRIEF SUMMARY [0006] The current disclosure describes the disruption of the genes encoding FS-7- associated surface antigen (FAS), FAS ligand (FASL), or both in pluripotent stem cells (PSCs) and other stem/progenitor cells followed by differentiation to T cells to generate T cells which are deficient in expression of FAS, FASL, or both. The disclosure provides for a cell comprising an inactivating modification in the FAS and/or FASL gene; wherein the cell does not express a functional FAS or FASL protein, respectively. The disruption of expression of FAS, FASL or both in mature cells enhances the survival and anti-tumor efficacy of the mature cells. Also described is a method for making a cell, the method comprising culturing a cell of the disclosure under conditions suitable for the in vitro differentiation of the cell into a mature cell. Also provided are cells produced by the methods of the disclosure as well as a method for treating a subject comprising administering cells of the disclosure. [0007] The FAS gene and/or FASL gene may be modified using gene editing to generate inactive FAS and/or FASL genes. The FAS and/or FASL gene are modified by contacting the cells with a site-specific Cas nuclease and a guide RNA or other method of genomic editing. The gene editing may also include a donor DNA that introduces an inactivating modification into the gene. The donor DNA may also be excluded in the methods of the disclosure. [0008] The cell may further comprise a heterologous nucleic acid encoding a transgene. The method may comprise or further comprise transferring a transgene into the cell. Transferring a transgene may comprise tranducing the cell with a nucleic acid encoding the transgene. The transgene may comprise a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). The cell may be one that expresses the transgene. The cell may be one that has constitutive or conditional expression of the transgene. The cell may be one that expresses the transgene in the mature T or NK cell. The transgene may comprise a T cell receptor (TCR). The TCR may be a tumor antigen-specific TCR, a virus-specific TCR, a cancer cell-specific TCR, a bacteria- specific TCR, or a cancer-testis antigen-specific TCR. The TCR may be a tumor antigenspecific TCR (i.e. a TCR that recognizes a tumor antigen). The TCR may be a virus antigenspecific TCR (i.e. a TCR that recognizes a viral antigen). The CAR may be a tumor antigenspecific CAR, virus-specific CAR, xeno-specific CAR, or bacteria-specific CAR, for example. The CAR may be a tumor antigen-specific CAR (i.e. a CAR that recognizes a tumor antigen. The transgene may be a transcription factor. Any one or more of these may be excluded in an an aspect described herein. The CAR may comprise or exclude an anti-CD19 CAR. The TCR may comprise or exclude an anti-NY-ESOl TCR. [0009] The transgene may be a cytokine receptor or cytokine. The transgene may exclude a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). The transgene may exclude a T cell receptor (TCR). The TCR may exclude a tumor antigen-specific TCR, a virus-specific TCR, a xeno-specific TCR, a cancer cell-specific TCR, a bacteria-specific TCR, or a cancer- testis antigen-specific TCR. The TCR may exclude a tumor antigen-specific TCR (i.e. a TCR