Search

US-20260125644-A1 - ENGINEERED IMMUNE CELLS EXPRESSING A CAR AND A PLURALITY OF PROTEIN EXPRESSION BLOCKERS AND USES THEREOF

US20260125644A1US 20260125644 A1US20260125644 A1US 20260125644A1US-20260125644-A1

Abstract

The present invention provides one or more expression vectors encoding one or more of an anti-CD7 chimeric antigen receptor (CAR), an anti-CD7 protein expression blocker, a kill gene or a suicide gene, and an anti-CD3 protein expression blocker. Also provided are immune cells comprising the one or more expression vectors. Also provided are methods of producing such one or more expression vectors or immune cells. Also provided are methods of using such one or more expression vectors or immune cells for treating cancer while reducing symptoms of Graft-versus-host disease (GvHD).

Inventors

  • Dario Campana
  • Ying Xim Tan
  • Alex Xing Fah WONG
  • Simin ZHENG

Assignees

  • MEDISIX THERAPEUTICS, INC.

Dates

Publication Date
20260507
Application Date
20230810

Claims (20)

  1. 1 .- 274 . (canceled)
  2. 275 . An engineered immune cell, comprising: (a) a first nucleic acid sequence encoding (i) a domain that binds to a subunit of a T cell receptor (TCR) complex linked to (ii) a synthetic localizing domain; (b) a second nucleic acid sequence encoding a chimeric antigen receptor (CAR); (c) a third nucleic acid sequence encoding a surface polypeptide binding domain linked to a synthetic surface polypeptide localizing domain; and wherein the engineered immune cell comprises at least twice as much of the first nucleic acid sequence as the second nucleic acid sequence or the third nucleic acid sequence.
  3. 276 . The engineered immune cell of claim 275 , wherein the subunit of the TCR complex is CD3ε.
  4. 277 . The engineered immune cell of claim 275 , wherein the synthetic localizing domain comprises an ER retention signal.
  5. 278 . The engineered immune cell of claim 277 , wherein the synthetic localizing domain further comprises a Myc tag.
  6. 279 . The engineered immune cell of claim 278 , wherein the first nucleic acid sequence comprises, in 5′ to 3′ direction, a sequence encoding the domain that binds to the subunit of the TCR complex, a sequence encoding the Myc tag, and a sequence encoding the ER retention signal.
  7. 280 . The engineered immune cell of claim 275 , wherein the second nucleic acid sequence and the third nucleic acid sequence are on the same nucleic acid molecule.
  8. 281 . The engineered immune cell of claim 280 , wherein the same nucleic acid molecule is a vector.
  9. 282 . The engineered immune cell of claim 281 , wherein the first nucleic acid sequence is on a nucleic acid molecule separate from the second nucleic acid sequence or the third nucleic acid sequence, and wherein the nucleic acid molecule having the first nucleic acid sequence is a first expression vector.
  10. 283 . The engineered immune cell of claim 275 , further comprising a fourth nucleic acid sequence encoding a kill gene.
  11. 284 . The engineered immune cell of claim 283 , wherein the kill gene comprises CD20 or a derivative thereof.
  12. 285 . The engineered immune cell of claim 275 , wherein the CAR comprises a target binding domain that binds to the surface polypeptide.
  13. 286 . The engineered immune cell of claim 285 , wherein the surface polypeptide binding domain is a first antibody or antigen binding domain thereof, and the target binding domain is a second antibody or antigen binding domain thereof.
  14. 287 . The engineered immune cell of claim 286 , wherein an amino acid sequence of the first antibody or antigen binding domain thereof and an amino acid sequence of the second antibody or antigen binding domain thereof are at least 80% identical.
  15. 288 . The engineered immune cell of claim 275 , wherein the surface polypeptide is CD7.
  16. 289 . The engineered immune cell of claim 275 , wherein the synthetic localizing domain or the synthetic surface polypeptide localizing domain comprises an endoplasmic reticulum (ER) retention sequence, a Golgi retention sequence, a proteasome localizing sequence, or a transmembrane domain.
  17. 290 . The engineered immune cell of claim 289 , wherein the synthetic localizing domain and the synthetic surface polypeptide localizing domain comprise a same ER retention sequence.
  18. 291 . The engineered immune cell of claim 275 , wherein a surface expression of the TCR complex and a surface expression of the surface polypeptide are downregulated in the engineered immune cell.
  19. 292 . The engineered immune cell of claim 275 , wherein the engineered immune cell is a T cell or a natural killer (NK) cell.
  20. 293 . The engineered immune cell of claim 275 , wherein the engineered immune cell is an allogeneic cell.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Stage Entry of PCT/US2023/072042, filed Aug. 10, 2023, which claims the benefit of U.S. Provisional Application Ser. No. 63/397,067, filed Aug. 11, 2022, U.S. Provisional Application Ser. No. 63/408,228, filed Sep. 20, 2022, and U.S. Provisional Application Ser. No. 63/425,174, filed Nov. 14, 2022, the entire content of each of which is incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 18, 2025, is named 62190_725_831_SL.txt and is 207,046 bytes in size. BACKGROUND OF THE INVENTION Chimeric antigen receptors (CARs) can redirect immune cells to specifically recognize and kill tumor cells. CARs are artificial multi-domain proteins constituted by a single-chain variable region (scFv) of an antibody linked to a signaling molecule via a transmembrane domain. When the scFv ligates its cognate antigen, signal transduction is triggered, resulting in tumor cell killing by CAR-expressing cytotoxic T lymphocytes (Eshhar Z, Waks T, et al. PNAS USA. 90(2):720-724, 1993; Geiger T L, et al. J Immunol. 162(10):5931-5939, 1999; Brentjens R J, et al. Nat Med. 9(3):279-286, 2003; Cooper L J, et al. Blood 101(4):1637-1644, 2003; Imai C, et al. Leukemia. 18:676-684, 2004). Clinical trials with CAR-expressing autologous T lymphocytes have shown positive responses in patients with B-cell refractory leukemia and lymphoma (see, e.g., Till B G, et al. Blood 119(17):3940-3950, 2012; Maude S L, et al. N Engl J Med. 371(16):1507-1517, 2014). The development of CAR technology to target T cell malignancies has lagged far behind the progress made for their B-cell counterparts. Novel therapies for T-cell malignancies are needed but progress to date has been slow. In particular, effective immunotherapeutic options are lacking and treatment of T-cell acute lymphocytic leukemia (T-ALL) relies on intensive chemotherapy and hematopoietic stem cell transplant. Despite aggressive treatment regimen associated with significant morbidity, results with these approaches are far from satisfactory. CAR-T cells have recently been developed in which the target antigen of the CAR-T is itself expressed in the CAR-T cell (Png et al., Blood, 2017, 1(25):2348-2360, WO 2018/098306). To avoid self-killing (e.g., fratricide), the CAR-T cells also express a PEBL that serves to reduce the expression of the target antigen on the cell surface of the CAR-T. To produce viable CAR-T cells, a protein expression blocker (PEBL) protein can be expressed to bind and sequester the target protein prior to the subsequent expression of the CAR. Due to the pre-existing presence of the target antigen on the cell surface of the resulting engineered T cells, simultaneous expression of the CAR and the PEBL may result in fratricide. In particular, the pre-existing cell surface target antigens may not be susceptible to sequestration by the newly expressed PEBL proteins, and may be recognized and targeted by the newly expressed CAR proteins. An alternative to simultaneous expression can be sequential expression. However, sequential expression of a PEBL and then a CAR in a T-cell creates several challenges for the clinical implementation of PEBL CAR-T cells. First, sequential engineering of the T cells requires the separate manufacture and administration of distinct viral vectors, one for the PEBL and a second for the CAR. This increases cost and time, as well as the complexity of experimental manipulation to produce the engineered CAR-T cells. In addition, sequential engineering of the T cells results in a complex mix of engineered cells in the final clinical product, creating challenges with product characterization, uniformity and efficacy. Because only a fraction of the T cells integrates the introduced gene at each engineering step, the final product (the engineered T cells) will comprise some cells that only received the PEBL gene, some cells that only received the CAR gene, and some cells that received both genes. In summary, there is a significant unmet need for new therapeutic options for patients with T-cell malignancies. There is a need for methods for producing an engineered CAR-T cell and eliminating CAR-mediated self-killing or fratricide of the T cell. Additionally, expression of endogenous T-cell receptors (TCRs) carries the risk for graft-versus-host-disease (GvHD), and a method for reducing risk of developing GvHD is needed for effective allogeneic CAR-T cell therapies. SUMMARY OF THE INVENTION Recognized herein is a need for improved CAR-T cell therapies and methods of producing the engineered CAR-T cells. The compositions and methods provided herein can produce engineered CAR-T cells and eliminate CAR-mediated self-killing or fratricide of the T cells. The compositions and methods pro