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US-12618048-B2 - Dual CAR-T cells

US12618048B2US 12618048 B2US12618048 B2US 12618048B2US-12618048-B2

Abstract

The present invention concerns new engineered immune cells expressing two CARs directed against two different targets, polynucleotides for preparing said immune cells, pharmaceutical compositions comprising said immune cells, and the use of said immune cells in the treatment of cancers.

Inventors

  • André Choulika
  • Laurent Poirot
  • Beatriz ARANDA ORGILLES
  • Philippe Duchateau

Assignees

  • CELLECTIS S.A.

Dates

Publication Date
20260505
Application Date
20210730
Priority Date
20200731

Claims (20)

  1. 1 . A genetically engineered immune cell expressing a Chimeric Antigen Receptor (CAR) specific for CD22 (CAR22) and a Chimeric Antigen Receptor specific for CD20 (CAR20) at its cell surface, wherein said CAR22 and said CAR20 are encoded by an exogenous nucleic acid incorporated in the genome of said immune cell, and wherein said exogenous nucleic acid comprises, from 5′ to 3′: i) a promoter, ii) a nucleic acid encoding said CAR20, iii) a nucleic acid encoding a self-cleaving peptide, and iv) a nucleic acid encoding said CAR22, whereby the same promoter controls the expression of said CAR20 and said CAR22, and wherein said exogenous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 32.
  2. 2 . The genetically engineered immune cell of claim 1 , wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, wherein the leader amino acid sequence set forth in SEQ ID NO: 1 is removed when said CAR20 and said CAR22 are at the cell surface.
  3. 3 . The genetically engineered immune cell of claim 1 , wherein said engineered immune cell is selected from the group consisting of a T-cell, a NK-cell, and a macrophage.
  4. 4 . The genetically engineered immune cell of claim 1 , wherein said engineered immune cell is a T-cell.
  5. 5 . The genetically engineered immune cell of claim 4 , wherein said engineered immune cell is a T-cell that is TCR negative.
  6. 6 . The genetically engineered immune cell of claim 4 , wherein said engineered T-cell has at least one allele encoding TCR alpha, TCR beta, and/or CD3 that has been inactivated by mutation.
  7. 7 . The genetically engineered immune cell of claim 4 , wherein said engineered T-cell has at least one inactivated allele, wherein said at least one inactivated allele is selected from the group consisting of β2m, PD1, CTLA4, dCK, CD52, GR, and a combination thereof.
  8. 8 . The genetically engineered immune cell of claim 1 , wherein said engineered cell expresses no further CAR than said CAR22 and CAR20.
  9. 9 . A pharmaceutical composition comprising the genetically engineered immune cell of claim 1 , and a pharmaceutically acceptable excipient.
  10. 10 . An isolated polynucleotide comprising, from 5′ to 3′: a) a promoter that controls the expression of a CAR20, followed by a nucleic acid encoding said CAR20; and b) a nucleic acid encoding a CAR22, wherein said nucleic acids of a) and b) are on a single nucleic acid molecule, and wherein a nucleic acid sequence encoding a self-cleaving peptide is located between said nucleic acids of a) and b), and wherein said single nucleic acid molecule comprises the sequence of SEQ ID NO: 32.
  11. 11 . The polynucleotide of claim 10 , wherein said polynucleotide does not comprise a nucleic acid encoding a further CAR than said CAR22 and CAR20.
  12. 12 . A vector comprising the isolated polynucleotide of claim 10 .
  13. 13 . A method of preparing the genetically engineered immune cell of claim 1 , said method comprising introducing into an immune cell the polynucleotide of claim 10 , or the vector of claim 12 .
  14. 14 . A method of treating a patient having a cancer associated with CD20 expression, CD22 expression, or CD20 expression and CD22 expression, said method comprising administering to said patient an effective amount of the genetically engineered immune cell of claim 1 .
  15. 15 . The method of claim 14 , wherein said cancer is selected from the group consisting of lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphocytic cancer, and acute myeloid leukemia (AML).
  16. 16 . The method of claim 14 , wherein said cancer is non-Hodgkin lymphoma or acute lymphocytic leukemia.
  17. 17 . The genetically engineered immune cell of claim 1 , wherein each of said CAR22 and said CAR20 do not comprise a leader sequence when said CAR22 and said CAR20 are at the cell surface.
  18. 18 . A genetically engineered immune cell expressing a Chimeric Antigen Receptor (CAR) that specifically binds to CD20 (CAR20) and a CAR that specifically binds to CD22 (CAR22) at its cell surface, wherein: (a) said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, optionally without the leader sequence of SEQ ID NO: 1; (b) said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, optionally without the leader sequence of SEQ ID NO: 1; (c) said genetically engineered immune cell comprises at least one allele encoding TCR alpha, TCR beta, or CD3 that has been inactivated; (d) said genetically engineered immune cell has at least one inactivated CD52 allele; and (e) said CAR20 and said CAR22 are encoded by an exogenous nucleic acid incorporated in the genome of said genetically engineered immune cell, wherein said exogenous nucleic acid comprises SEQ ID NO: 32.
  19. 19 . The genetically engineered immune cell of claim 18 , wherein said genetically engineered immune cell comprises at least one allele encoding TCR alpha that was inactivated by mutation.
  20. 20 . A population of T-cells comprising the genetically engineered immune cell of claim 18 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a National Stage application under 35 U.S.C. § 371 of International Application No. PCT/EP2021/071400, having an International Filing Date of Jul. 30, 2021, which claims benefit of priority to DK Application Serial No. PA202070509, filed Jul. 31, 2020. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named “47246-0013US1.txt.” The ASCII text file, created on Jul. 28, 2021, is 110,191 bytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety. FIELD The present invention relates to the field of cell immunotherapy and more particularly to new engineered immune cells expressing two CARs directed against two different targets, useful in the treatment of cancers. BACKGROUND Approximately every 3 minutes one person in the United States (US) is diagnosed with a blood cancer. An estimated combined total of 178,520 people in the US are expected to be diagnosed with leukemia, lymphoma or myeloma in 2020. New cases of leukemia, lymphoma and myeloma are expected to account for 9.9 percent of the estimated 1,806,590 new cancer cases diagnosed in the US in 2020 (Cancer Facts & Figures, 2020. American Cancer Society). The development of chimeric antigen receptor (CAR) T-cell therapy for hematological malignancies represents one of the most remarkable therapeutic advances in the past decade (Holstein et al, 2020, Clin. Pharmacol. Ther. 107(1): 112-122). Indeed, as a rapidly progressing field in oncology, the adoptive transfer of CAR-T cells has shown striking efficacy in the management of hematological malignancies and has been reported in a number of clinical trials. Chimeric antigen receptors (“CAR”) expressing immune cells are cells which have been genetically engineered to express CARs usually designed to recognize specific tumor antigens and kill the cancer cells that express these tumor antigens. It is not excluded that the CAR immune cells can activate the immune system to eliminate tumors. These are generally T cells expressing CARs (“CAR-T cells”) or Natural Killer cells expressing CARs (“CAR-NK cells”) or macrophages expressing CARs. CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signalling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain derived from a monoclonal antibody, consisting of a single chain variable fragment (scFv), which contains the light and heavy variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signalling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signalling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010, Blood 116(7):1035-44). Adoptive immunotherapy, which involves the transfer of autologous or allogeneic antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer as confirmed by the increase in the number of CAR-T cells approved by the US Food and Drug Administration (FDA) (e.g. Novartis' anti-CD19 CAR-T tisagenlecleucel (Kymriah™) for the treatment of precursor B-cell acute lymphoblastic leukemia, Kite Pharma's anti-CD19 CAR-T axicabtagene ciloleucel (Yescarta™) for certain types of large B-cell lymphoma in adult patients). Despite the progress in research and development of CAR-T cell therapy, there still remains a need for improved CAR-T cells which could target a wider range of cancers as well as recurrent cancers and/or cancers for which the expression of the cancer-associated antigens are very variable and evolve with the time or during or after the treatments. SUMMARY The inventors have developed new CAR-T cells targeting CD20 and CD22 antigens, which can be activated by the tumor cells expressing CD20 and CD22 at variable levels, and constitute an improvement over CAR-T cells of the prior art. A first aspect relates to a genetically engineered immune cell expressing a Chimeric Antigen Receptor (CAR) specific for CD22 (CAR22) and a Chimeric Antigen Receptor specific for CD