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US-12618066-B2 - Promoter having high activity in activated T-cell

US12618066B2US 12618066 B2US12618066 B2US 12618066B2US-12618066-B2

Abstract

Provided is a promoter having high activity in an activated T-cell. The promoter comprises, from 5′-end to 3′-end, a CMV enhancer, an IFNγ promoter, and a long terminal repeat sequence from human T-cell leukemia virus that are connected in sequence. The promoter exhibits greater activity in an activated immune cell than the existing promoters and is low in activity or inactive in other non-immune cells.

Inventors

  • Tao Liu
  • Yuan Fang
  • Haixia GAO
  • Qijun Qian

Assignees

  • Shanghai Cell Therapy Group Co., Ltd.

Dates

Publication Date
20260505
Application Date
20201113
Priority Date
20191115

Claims (18)

  1. 1 . A promoter comprising, from 5′-end to 3′-end, a CMV enhancer having the nucleotide sequence of SEQ ID NO: 8, an IFNY promoter having the nucleotide sequence of SEQ ID NO: 4, and a long terminal repeat sequence from human T-cell leukemia virus having the nucleotide sequence of SEQ ID NO: 3, which are connected in sequence.
  2. 2 . A nucleic acid construct, wherein the nucleic acid construct contains the promoter according to claim 1 , and a gene of interest operably linked to the promoter.
  3. 3 . The nucleic acid construct according to claim 2 , wherein the gene of interest encodes an autocrine antibody and/or a cytokine.
  4. 4 . The nucleic acid construct according to claim 2 , wherein the nucleic acid construct is an expression cassette.
  5. 5 . A vector, wherein the vector contains the promoter according to claim 1 , or a nucleic acid construct comprising the promoter.
  6. 6 . The vector according to claim 5 , wherein the vector is an expression vector or a cloning vector.
  7. 7 . An isolated host cell, wherein the host cell contains the promoter according to claim 1 , or a nucleic acid construct containing the promoter, or a vector containing the promoter or the nucleic acid construct.
  8. 8 . The isolated host cell according to claim 7 , wherein: the isolated host cell is an immune cell with its genome integrated with the nucleic acid construct; the isolated host cell is an immune cell containing: the promoter and a coding sequence of cytokine operably linked to the promoter, and/or the promoter and a coding sequence of an immune checkpoint antibody or its bispecific antibody operably linked to the promoter; or the isolated host cell is an immune cell with its genome integrated with: an expression cassette containing the promoter and a coding sequence of cytokine operably linked to the promoter, and/or an expression cassette containing the promoter and a coding sequence of an immune checkpoint antibody or its bispecific antibody operably linked to the promoter.
  9. 9 . The isolated host cell according to claim 8 , wherein the immune cell further expresses a chimeric antigen receptor (CAR) or has an expression vector of CAR.
  10. 10 . The isolated host cell according to claim 8 , wherein, the immune checkpoint antibody is selected from the group consisting of: PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody; or the cytokine is selected from the group consisting of: interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemokine and growth factor.
  11. 11 . The nucleic acid construct according to claim 3 , wherein the autocrine antibody is an alpaca-derived VHH antibody and/or is an immune checkpoint antibody, or the cytokine is selected from the group consisting of: interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemokine and growth factor.
  12. 12 . The nucleic acid construct according to claim 11 , wherein the immune checkpoint antibody is selected from a group consisting of PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody.
  13. 13 . The host cell according to claim 8 , wherein the immune cell is a T cell.
  14. 14 . A method for expressing a protein of interest in a cell of interest or improving expression of a gene of interest in an activated immune cell, comprising: transferring into the cell of interest a nucleic acid molecule containing a coding sequence of the protein of interest that is operably linked to the promoter according to claim 1 and culturing the cell under a condition that allows expression of the protein of interest; or transferring into the activated immune cell a vector containing the gene of interest which is operably linked to the promoter and culturing the activated immune cell under a condition suitable for the expression of the gene of interest.
  15. 15 . The method according to claim 14 , wherein the gene of interest encodes an autocrine antibody and/or a cytokine.
  16. 16 . The method according to claim 14 , wherein: the autocrine antibody is an alpaca-derived VHH antibody and/or is an immune checkpoint antibody; and/or the cytokine is selected from the group consisting of: interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemokine and growth factor.
  17. 17 . The method according to claim 16 , wherein the autocrine antibody is selected from a group consisting of antibody PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody.
  18. 18 . The method according to claim 14 , wherein during culturing an anti-CD28 antibody and an optional immunogen are used for stimulating the cell of interest or the activated immune cell.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a national stage application under 35 U.S.C. § 371 of International Application No. PCT/CN2020/128526, filed internationally on Nov. 13, 2020, which claims priority to Chinese Application No. 201911120441.4, filed Nov. 15, 2019. SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 699532000600SeqList.txt, date created: May 12, 2022, size: 12,984 bytes). TECHNICAL FIELD The invention relates to a promoter having high activity in activated T cells. BACKGROUND Promoter is a component of gene, usually located upstream of the 5′ end of a structural gene. It is a DNA sequence recognized, bound, and transcribed first by RNA polymerase. Promoter is one of the important factors affecting the efficiency of transgenic expression. The selection of efficient promoter is the key to the efficient expression of foreign genes. According to the transcription pattern of promoters, they can be divided into three categories: constitutive promoters, tissue or organ specific promoters and inducible promoters. A constitutive promoter refers to that there is no significant difference in gene expression in different tissues, organs and development stages under the regulation of the constitutive promoter, so it is called constitutive promoter. Constitutive promoters commonly used in mammals include those derived from virus: mouse or human cytomegalovirus (CMV) promoters (MCMV and HCMV respectively), monkey vacuolar virus SV40 promoter; and those naturally derived from human genome: EF1α promoter, ubiquitin promoter (Ubi), β-Actin promoter, PGK-1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, eIF4A1 promoter, EGR1 promoter, FerH promoter, SM22α promoter, Endothelin-1 promoter, etc. In tumor immunotherapy, it is important to maintain the efficient and stable expression of foreign genes. However, some virus derived constitutive promoters are easy to be turned off due to epigenetic modification despite their high transient expression activity (such as CMV promoter). Although the expression of some human natural constitutive promoters or tumor specific promoters is stable, their expression activity is relatively weak, which is difficult to meet the needs of immunotherapy. Therefore, the researchers designed and constructed a series of artificial chimeric promoters, which contain some cis regulatory elements, mainly including the promoter core sequence that can express stably, and the upstream enhancer or downstream intron that can enhance the expression efficiency. The representative is the chimeric promoter CAG (including human CMV enhancer chicken β-actin promoter rabbit β-globin intron), which is widely used in the expression of foreign genes. Enhancers are DNA sequences that increase the transcription frequency of genes linked to them. Enhancers increase the transcription of downstream genes through promoters. Effective enhancers can be located at the 5′ end of the gene, at the 3′ end of the gene, and some can also be in the intron of the gene. The effect of enhancer is obvious. Generally, it can increase the gene transcription frequency by 10-200 times, and some can even be as high as thousands of times. SUMMARY OF INVENTION The invention constructs a promoter is composed of a CMV enhancer, an IFNγ promoter, and a long terminal repeat (LTR) sequence from HTLV (human T-cell leukemia virus). The promoter exhibits greater activity in an activated immune cell than the existing promoters and is low in activity or inactive in other non-immune cells. Therefore, the invention provides a promoter comprising, from 5′-end to 3′-end, a CMV enhancer, an IFNγ promoter, and a long terminal repeat sequence from human T-cell leukemia virus that are connected in sequence. In one or more embodiments, the CMV enhancer is selected from the group consisting of: a CMV enhancer having the nucleotide sequence shown in SEQ ID NO: 8, or a CMV enhancer from human CMV having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 8. In one or more embodiments, the IFNγ promoter is selected from the group consisting of: the IFNγ promoter having the nucleotide sequence shown in SEQ ID NO: 4, or IFNγ promoter from human having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 4. In one or more embodiments, the long terminal repeat sequence from the human T-cell leukemia virus is selected from the group consisting of: a long terminal repeat sequence having the nucleotide sequence shown in SEQ ID NO: 3, or a long terminal repeat sequence from the human T-cell leukemia virus having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the invention also provides a nucleic acid molecule with its base sequence complementary to the