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US-12617828-B2 - Genetically modified immune cell, preparation method therefor, and application

US12617828B2US 12617828 B2US12617828 B2US 12617828B2US-12617828-B2

Abstract

Provided are a genetically modified immune cell, a preparation method therefor, and an application. The immune cell overexpresses HIL-6 and/or L-GP130. HIL-6 or L-GP130 continuous overexpression/conditionally induced overexpression in the immune cell reduces the side effects of CAR-T therapy while maintaining immune and anti-tumour effects, and has potential value in the treatment of malignant tumours and AIDS.

Inventors

  • Peng Li
  • Yao Yao
  • Zhiwu JIANG
  • Zhaoyang Tang
  • Rui Liao
  • Xiaohan Huang
  • Simiao Lin
  • Suna WANG
  • Youguo LONG
  • Qiting WU

Assignees

  • SHENZHEN IN VIVO BIOMEDICINE TECHNOLOGY LIMITED COMPANY

Dates

Publication Date
20260505
Application Date
20200506
Priority Date
20190403

Claims (11)

  1. 1 . An immune cell overexpressing HIL-6 and L-GP130, wherein the HIL-6 has an amino acid sequence of SEQ ID NO: 1, wherein the L-GP130 has an amino acid sequence of SEQ ID NO: 3, wherein the immune cell is a T cell and wherein the T cell is modified with a CAR molecule.
  2. 2 . The immune cell according to claim 1 ; the T cell is selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a CD4+CD8+ T cell, an NKT cell and a gamma delta (γδ) T cell.
  3. 3 . The immune cell according to claim 1 , wherein an antigen recognized by an extracellular scFv sequence of the CAR molecule comprises any one of 5T4, alpha-5 beta-1 (α5β1)-integrin, 707-AP, AFP, ART-4, B7H4, B7-H3, BAGE, beta (β)-integrin/m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, myosin/m, MUC1, MUM-1, MUM-2, MUM-3, PSCA, NA88-A, PAP, protease-3, GPC3, p190minor bcr-abl, Pml/RARR, PRAME, PSA, PSM, PSMA, RAGE, RU1, RU2, SAGE, SART-1, SART-3, survivin, TEL/AML1, PD-1, PD-L1, CTLA-4, TIM3, LAG3, TGF3, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, IL-13RIN, CD123, GUCY2C, NY-Eso-1 or NY-Eso-B.
  4. 4 . The immune cell according to claim 3 , wherein an scFv of the anti-CD 19 CAR has an amino acid sequence of SEQ ID NO: 5 and a nucleic acid sequence of SEQ ID NO: 6 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12; an scFv of the anti-GPC3 CAR has an amino acid sequence of SEQ ID NO: 13 and a nucleic acid sequence of SEQ ID NO: 14; an scFv of the anti-MUC1 CAR has an amino acid sequence of SEQ ID NO: 15 and a nucleic acid sequence of SEQ ID NO: 16; an scFv of the anti-Mesothelin has an amino acid sequence of SEQ ID NO: 17 and a nucleic acid sequence of SEQ ID NO: 18; an scFv of the anti-PSCA has an amino acid sequence of SEQ ID NO: 19 and a nucleic acid sequence of SEQ ID NO: 20; an scFv of the anti-HER2 has an amino acid sequence of SEQ ID NO: 21 and a nucleic acid sequence of SEQ ID NO: 22.
  5. 5 . The immune cell according to claim 1 , wherein the CAR molecule has an intracellular co-stimulatory signal transduction domain comprising an intracellular domain of any one of molecules of CD28, 4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, DAP10, CD27, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1, CD2, CD7, LIGHT, NKG2C, NKG2D, NKp46, NKp30, NKp44, DNAM1, B7-H3, and CD83, or comprising a combination of intracellular domains of at least two of these molecules.
  6. 6 . The immune cell according to claim 5 , wherein the TLR2 has an amino acid sequence of SEQ ID NO: 23 and a nucleic acid sequence of SEQ ID NO: 24; the 4-1BB has an amino acid sequence of SEQ ID NO: 25 and a nucleic acid sequence of SEQ ID NO: 26; the CD28 has an amino acid sequence of SEQ ID NO: 27 and a nucleic acid sequence of SEQ ID NO: 28; the TLR1 has an amino acid sequence of SEQ ID NO: 29 and a nucleic acid sequence of SEQ ID NO: 30; the DAP10 has an amino acid sequence of SEQ ID NO: 31 and a nucleic acid sequence of SEQ ID NO: 32; a signal peptide of the CAR molecule has a nucleic acid sequence of SEQ ID NO: 33; a transmembrane region of the CAR molecule has a nucleic acid sequence of SEQ ID NO: 34; CD32 of the CAR molecule has a nucleic acid sequence of SEQ ID NO: 35.
  7. 7 . A method for preparing the immune cell according to claim 1 , comprising overexpressing HIL-6 and L-GP130 in an immune cell, wherein the HIL-6 has an amino acid sequence of SEQ ID NO: 1, wherein the L-GP130 has an amino acid sequence of SEQ ID NO: 3, wherein the immune cell is a T cell and wherein the T cell is modified with one or two of a CAR molecule.
  8. 8 . A pharmaceutical composition, comprising the immune cell according to claim 1 ; the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, an excipient or a diluent.
  9. 9 . A method for the treatment of tumors comprising: administering the immune cell of claim 1 ; wherein the tumor comprises CD19+B-cell acute lymphoblastic leukemia, MUC1+ lung cancer, or GPC3+ liver cancer.
  10. 10 . The immune cell according to claim 1 , wherein the T cell has a nucleic acid sequence of SEQ ID NO: 2.
  11. 11 . The immune cell according to claim 1 , wherein the T cell has a nucleic acid sequence of SEQ ID NO: 4.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/CN2020/088806, filed May 6, 2020, which claims the benefit of priority under 35 U.S.C. § 119 (e) to CN Application No: 201910267702.9, filed Apr. 3, 2019 and CN application No. 201910372692.5, filed May 6, 2019, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 6, 2021 is named 51766-502N01US ST25_Sequence.txt and is 88 KB in size. TECHNICAL FIELD The present application belongs to the field of biotechnology, relates to a genetically modified immune cell, a preparation method therefor, and use thereof, and in particular, relates to an immune cell overexpressing HIL-6 and/or L-GP130, a preparation method therefor, and use thereof. BACKGROUND The clinical application of anti-CD19 chimeric antigen receptor (CAR) T cells (tisagenlecleucel and axicabtagene ciloleucel) has shown that such cells have a significant killing effect on CD19 positive B-cell malignant tumors. However, there are still many problems in CAR T cell therapy. In the treatment of leukemia, the CAR T cell therapy has the problem of a high recurrence rate, and the promotion of the maintenance of CAR T cells and the formation of memory T cells may be the technical breakthrough to solve this problem. In addition, the results of clinical treatment of anti-CD19 CAR T cells show that there is a positive correlation between the response rate of patients to the CAR T cell therapy and the occurrence of cytokine release syndrome (CRS), and CRS may lead to side effects such as CAR-T cell-related encephalopathy syndrome (CRES), which seriously limits the clinical application of CAR T cells. In the treatment of solid tumors, CAR T cells have a poor curative effect, and no related clinical research breakthrough has been reported yet. The activation and expansion of T cells are required for effective coordination signals, including T-cell receptor (TCR) signals (first signal), co-stimulatory signals (second signal), and cytokine signals (third signal). Molecules carrying the third signal (such as IL-12, IL-7, and IL-15) significantly improve the expansion, migration, persistence, and anti-tumor efficacy of CAR T cells. CRS is detected with interleukin 6 (IL-6) as the indicator. As a pleiotropic cytokine in the family of chemokines, IL-6 is mainly generated in mononuclear macrophages, Th2 cells, vascular endothelial cells, and fibroblasts. IL-6 has many target cells and complex biological effects. IL-6 can trigger a wide range of cellular and physiological reactions, including immune response, inflammation, hematopoiesis, and tumorigenesis, by regulating growth, gene activation, proliferation, survival, and differentiation of cells. IL-6 forms a protein complex by binding to non-signal transducer IL-6Rα, then dimerizes with subunit glycoprotein 130 (GP130), and initiates signal transduction cascades through transcription factors, Janus kinases (JAKs), signal transducers, and transcription activators (STATs). The binding of IL-6 and IL-6R includes membrane-binding IL-6R (mIL-6R) and soluble IL-6R (sIL-6R), which are called classical signal transduction and nonclassical signal transduction, respectively. In the existing art, the effect of IL-6 is usually inhibited by using IL-6 monoclonal antibody (PMID: 27049586) or IL-1 beta monoclonal antibody (PMID: 29808007 and PMID: 29808005), or the side effects of CAR T cell therapy are inhibited by targeting IL-6 and IL-6R (EP3305890A1, US20180369282A1, U.S. Ser. Nos. 15/018,797, and 15/106,657). However, the mechanism of action of IL-6 is complex and has two sides. One-sided inhibition of IL-6 may affect the anti-tumor effect of CAR T cells. WO2017007985 discloses a method for treating cancer using IL-6. However, the treatment with IL-6 alone may activate both membrane-binding IL-6R (mIL-6R) and soluble IL-6R (sIL-6R). mIL-6R is mostly related to the non-tumor killing. Excessive activation of mIL-6R may interfere with the function of immune cells and consume the amount of IL-6 in soluble IL-6R. Early reports have shown that IL-6 has anti-tumor effects in mouse models, but mouse models have limited the discovery of side effects of IL-6. With the development of science and technology, researchers have gradually realized that IL-6 can not only cause serious side effects, but also cause inflammatory microenvironment, trigger cytokine storm in patients, and promote tumor growth or immune escape when IL-6 is used systemically. In recent study, the side effects of IL-6 are often blocked by using IL-6 receptor monoclonal antibody and knocking out IL-6 receptor (PMID: 27076371). However, blocking IL-6 will affect the maximization of the anti-tumor