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CD7-CAR-T cell as well as preparation method and application thereof

A cell and antigen technology, applied in the field of CD7-CAR-T cells and their preparation, can solve the problems of knocking out CD7 molecules, weakened antigen-antibody binding force and stability, and long-term existence, achieving significant killing effect and avoiding self-contrast. The effect of killing and ensuring safety

Active Publication Date: 2022-05-31
ADVANCED BIOTHERAPEUTICS LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a lot of clinical data have been accumulated, the role of autologous bone marrow transplantation in the treatment of acute leukemia is still controversial due to the wide differences in the curative effects of various reports. Rate (LFS) varies from less than 30% to over 70%
[0008]Compared with CAR-T therapy for B-ALL, CD7-CAR-T cells are used to target T-cell acute lymphoblastic leukemia and CD7-positive lymphoma, etc. Still facing great technical problems, this is because both normal effector T cells and T cell tumors express the CD7 antigen, which will lead to the "cannibalistic" effect of CD7-CAR-T cells. Therefore, CD7-CAR-T Cells are difficult to produce successfully in vitro; in clinical applications, it is also necessary to consider that CD7-CAR-T cells cannot be allowed to exist in vivo without restriction for a long time to avoid serious side effects
At present, there are only a few studies on CD7-CAR-T technology. In order to avoid the "cannibalism" effect, the method of CRISPR gene editing is used to knock out the CD7 gene in T cells, but it is difficult to knock out the CD7 molecule 100% in practice and theory , while the risk of clinical application of graft-versus-host disease (GVHD) remains
At the same time, the possible side effects caused by the natural "off-target effects" of gene editing will also limit the widespread development of clinical applications
The use of nano-body (nanobody) technology to reduce the volume of the antibody-binding region has certain advantages, but the disadvantage is that the reduced and single-armed antibody will lead to relatively weakened antigen-antibody binding and stability, resulting in CD7-CAR- Decreased T cell potency

Method used

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  • CD7-CAR-T cell as well as preparation method and application thereof
  • CD7-CAR-T cell as well as preparation method and application thereof
  • CD7-CAR-T cell as well as preparation method and application thereof

Examples

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preparation example Construction

[0090] The present invention provides the preparation method of the immune effector cells, which includes infecting the immune effector cells with the isolated nucleic acid or the vector of the present invention. Preferably, the present invention produces genetically engineered immune effector cells by introducing chimeric antigen receptors into immune effector cells, such as T cells.

[0091] It should be noted that methods for introducing nucleic acid or vector into mammalian cells are known in the art, and the vector can be transferred into immune effector cells by physical, chemical or biological methods. Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Chemical means for introducing nucleic acids or vectors into immune effector cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, b...

Embodiment 1

[0107] This example is for the preparation of mouse monoclonal antibody against CD7 antigen.

[0108] In this example, 5 polypeptides were designed and synthesized for the CD7 antigen to immunize BALB / c mice respectively. After cell fusion, primary screening, and secondary screening, a positive clone was obtained that simultaneously recognized the polypeptide BST001-2 and CD7 recombinant protein. The clone number of the positive hybridoma cell line is 5B5.

[0109] The amino acid sequence of the CD7 antigen precursor protein is shown in SEQ ID NO: 1, wherein amino acid residues 26-180 are the extracellular domain of the CD7 antigen. The CD7 recombinant protein is a recombinant human CD7 protein (with a his tag, a product of Biopsies, the article number is 11028-H08H), and its amino acid sequence is the sequence of the extracellular domain of the CD7 antigen, as shown in SEQ ID NO:2.

[0110] The amino acid sequences of the five synthetic antigen polypeptides used to immunize ...

Embodiment 2

[0153] This example is for the construction of CD7-Blocker and CD7-CAR lentiviral expression vectors.

[0154] The CD7 blocking molecules CD7-Blocker and CD7-CAR were respectively constructed into the third-generation lentiviral expression plasmids by conventional technical means in the field. The plasmid is pCDH-EF1(X6)-MCS-T2A-Puro, and its map is as follows figure 1 As shown, the vector linearization restriction sites are XbaI and SalI, and the DNA sequences of CD7-Blocker and CD7-CAR (including the N-terminal KOZAC sequence) are inserted between the two restriction sites. The molecular structure of CD7-Blocker is as follows figure 2As shown, the CD7-Blocker molecule is composed of CD8a signal peptide SP, anti-CD7 scFv composed of (G4S)3 linker connecting VL and VH, and ER Retention Domain (endoplasmic reticulum localization domain). The front end of SP is inserted to promote expression The KOZAK sequence. Its full-length amino acid sequence is shown in SEQ ID NO: 18, a...

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Abstract

The invention discloses a CD7-CAR-T cell as well as a preparation method and application thereof. The CD7-CAR-T cell comprises an antibody targeting a CD7 antigen or an antigen binding fragment of the antibody, and the antibody or the antigen binding fragment of the antibody contains heavy chain variable regions of antigen complementary determining regions CDR1, CDR2 and CDR3 with amino acid sequences shown in SEQ ID NO.12-14; and a light chain variable region having antigen complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequences as shown in SEQ ID NO.15-17. According to the present invention, the antibody and the CD7-CAR based on the antibody fragment have strong affinity with the CD7 antigen molecule, and the blocking molecule can almost completely block the expression of the CD7 molecule on the cell surface without affecting the normal amplification of the T cell, such that the self-killing of the CD7-CAR-T cell can be effectively avoided. In addition, the CD7-CAR-T cell disclosed by the invention has a remarkable and specific killing effect on CD7 positive target cells, and a beneficial CAR-T cell is provided for clinical application of cell therapy.

Description

technical field [0001] The invention relates to the technical field of biological immunotherapy, in particular to CD7-CAR-T cells and their preparation methods and applications. Background technique [0002] Acute lymphocytic leukemia (ALL) is a common malignant tumor of the blood system, mainly originating from B lymphocytes and T lymphocytes. Primitive immature lymphocytes undergo multi-step specific gene damage to form tumor cells, which abnormally proliferate and accumulate in the bone marrow, and inhibit the hematopoietic function of normal bone marrow stem cells, leading to the occurrence of ALL. At the same time, these tumor cells can also infiltrate into extramedullary tissues (such as lymph nodes, meninges, liver, gonads, etc.), causing related tissue damage. [0003] ALL can occur in all age groups, and the incidence rate of children and young adults is higher at 3.6 / 100,000, while the incidence rate of adult ALL is lower at 0.69 / 100,000. Initial chemotherapy and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C12N15/86C12N5/0686A61K39/001129A61P35/00A61P35/02C07K2317/565C07K2317/56C07K2319/04C07K2319/03C07K2319/02C07K2319/74C07K2319/33C12N2740/15043C12N2510/00A61K2039/5158
Inventor 许中伟张海燕
Owner ADVANCED BIOTHERAPEUTICS LTD
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