Chimeric antigen receptor structure for enhancing killing activity of CAR-T cells and preparation method of chimeric antigen receptor structure

Abstract

The invention relates to a chimeric antigen receptor structure for enhancing killing activity of CAR-T cells and a preparation method, the chimeric antigen receptor structure comprises an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, the intracellular domain comprises double costimulatory factors CD27 and 4-1BB, and specifically comprises a continuous truncated functional domain fragment of GM-CSF protein and a chimeric antigen receptor; the continuous truncated functional domain fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a C helix fragment; the continuous truncated functional domain fragment of the GM-CSF protein is connected with the chimeric antigen receptor through a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region and a human CD3 zeta signal domain. The CAR structure provided by the invention adopts a double-costimulatory factor design, and the activation and durability of T cells are effectively improved, so that the killing activity of the CAR-T cells is remarkably enhanced. The preparation method is simple, efficient and suitable for large-scale production, and provides powerful support for clinical application of CAR-T cell therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and cell therapy, and in particular to a chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells and a preparation method thereof. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) therapy is a new type of tumor immunotherapy, which uses genetic engineering technology to introduce chimeric antigen receptors into T cells, enabling them to specifically recognize and kill tumor cells. Although CAR-T therapy has achieved remarkable results in the treatment of certain blood tumors, it still has some limitations, such as insufficient persistence of CAR-T cells in the body and unsatisfactory killing activity, which restricts its wider application.

[0003] The existing CAR-T cell construction technology is difficult to fully exert its maximum killing potential when dealing with complex tumor microenvironments. Therefore, the development of a chimeric antigen receptor structure and preparation method that can effectively enhance the killing activity of CAR-T cells has important clinical significance and application prospects. Summary of the invention

[0004] The purpose of the present invention is to provide a chimeric antigen receptor structure that enhances the killing activity of CAR-T cells and a method for preparing the structure, so as to improve the ability of CAR-T cells to recognize and kill tumor cells and improve the therapeutic effect of CAR-T therapy, thereby solving the technical problems mentioned above.

[0005] The solution of the present invention to solve the above technical problems is as follows: a chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells, the chimeric antigen receptor structure comprising an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, the intracellular domain comprising dual co-stimulatory factors CD27 and 4-1BB, specifically comprising a continuously truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor through a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

[0006] Furthermore, the extracellular domain is composed of a single-chain variable fragment (scFv) for recognizing and binding to tumor antigens.

[0007] Furthermore, the nucleotide sequence of the continuously truncated functional region fragments of the GM-CSF protein is shown in SEQ ID NO.1.

[0008] Furthermore, the chimeric antigen receptor structure also comprises an exon2 functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the exon2 functional region fragment is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3ζ signal domain.

[0009] Further, the hinge region connects the scFv and the transmembrane domain, providing the flexibility required for CAR expression and antigen binding.

[0010] Furthermore, the nucleotide sequence of the exon2 functional region fragment is shown as SEQ ID NO.1.

[0011] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0012] The purpose of the present invention is to provide a chimeric antigen receptor structure that enhances the killing activity of CAR-T cells and a method for preparing the structure, as follows: A method for preparing a chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells, comprising the steps of gene cloning, T cell collection and activation, gene transduction, in vitro amplification and screening, and preparation and reinfusion.

[0013] Furthermore, the gene transduction step utilizes a viral vector to transduce gene fragments encoding various domains of CAR into activated T cells.

[0014] The beneficial effects of the present invention are: the CAR structure of the present invention adopts a dual co-stimulatory factor design, which effectively improves the activation degree and persistence of T cells, thereby significantly enhancing the killing activity of CAR-T cells. In addition, the preparation method of the present invention is simple and efficient, suitable for large-scale production, and provides strong support for the clinical application of CAR-T cell therapy.

[0015] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1: Schematic diagram of the CAR structure of the present invention.

[0017] (The figure shows the extracellular domain, hinge region, transmembrane domain and intracellular domain, where the intracellular domain contains the dual co-stimulatory factors CD27 and 4-1BB.) Figure 2 : CAR-T cell killing activity detection results.

[0018] (The figure shows a comparison of the killing efficiency of tumor cells by the CAR-T cells of the present invention and the CAR-T cells of the control group. The results show that the CAR-T cells of the present invention have higher killing activity.) DETAILED DESCRIPTION The following is combined with Figure 1-2 The principles and features of the present invention are described, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. In the following paragraphs, the present invention is described in more detail by way of example with reference to the accompanying drawings. The advantages and features of the present invention will become clearer according to the following description and claims. It should be noted that the drawings are all in a very simplified form and are not in precise proportions, and are only used to facilitate and clearly assist in explaining the purpose of the embodiments of the present invention.

[0019] It should be noted that when a component is referred to as being "fixed to" another component, it may be directly on the other component or there may also be a component centered. When a component is considered to be "connected to" another component, it may be directly connected to the other component or there may also be a component centered. When a component is considered to be "set on" another component, it may be directly set on the other component or there may also be a component centered. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0021] A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells, the chimeric antigen receptor structure comprising an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, the intracellular domain comprising dual co-stimulatory factors CD27 and 4-1BB, specifically comprising a continuously truncated functional region fragment of a GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

[0022] Furthermore, the extracellular domain is composed of a single-chain variable fragment (scFv) for recognizing and binding to tumor antigens.

[0023] Furthermore, the nucleotide sequence of the continuously truncated functional region fragments of the GM-CSF protein is shown in SEQ ID NO.1.

[0024] Furthermore, the chimeric antigen receptor structure also comprises an exon2 functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the exon2 functional region fragment is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3ζ signal domain.

[0025] Further, the hinge region connects the scFv and the transmembrane domain, providing the flexibility required for CAR expression and antigen binding.

[0026] Furthermore, the nucleotide sequence of the exon2 functional region fragment is shown as SEQ ID NO.1.

[0027] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0028] The purpose of the present invention is to provide a chimeric antigen receptor structure that enhances the killing activity of CAR-T cells and a method for preparing the structure, as follows: A method for preparing a chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells, comprising the steps of gene cloning, T cell collection and activation, gene transduction, in vitro amplification and screening, and preparation and reinfusion.

[0029] Furthermore, the gene transduction step utilizes a viral vector to transduce gene fragments encoding various domains of CAR into activated T cells.

[0030] The beneficial effects of the present invention are: the CAR structure of the present invention adopts a dual co-stimulatory factor design, which effectively improves the activation degree and persistence of T cells, thereby significantly enhancing the killing activity of CAR-T cells. In addition, the preparation method of the present invention is simple and efficient, suitable for large-scale production, and provides strong support for the clinical application of CAR-T cell therapy.

[0031] Example 1: Construction of CAR gene expression plasmid 1. According to the CAR structure design of the present invention, gene fragments encoding each domain of CAR are synthesized.

[0032] 2. Clone these gene fragments into appropriate expression vectors (such as retroviral vectors or lentiviral vectors) to construct CAR gene expression plasmids.

[0033] 3. Sequencing verification of the constructed CAR gene expression plasmid to ensure that its sequence is correct.

[0034] Example 2: Preparation of CAR-T cells 1. Collect peripheral blood mononuclear cells from the patient and separate T cells using appropriate methods.

[0035] 2. Activate the isolated T cells to put them in a suitable proliferation state.

[0036] 3. Use a viral vector to transduce the CAR gene expression plasmid constructed in Example 1 into activated T cells.

[0037] 4. The transduced T cells are expanded in vitro, and T cells with high expression of CAR are screened by flow cytometry and other methods.

[0038] 5. The selected CAR-T cells are prepared into preparations and infused back into the patient's body for anti-tumor treatment.

[0039] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells, characterized in that: The chimeric antigen receptor structure includes an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, wherein the intracellular domain contains dual co-stimulatory factors CD27 and 4-1BB, specifically, a continuously truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein includes an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor includes a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

2. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 1, characterized in that: The extracellular domain is composed of a single-chain variable fragment (scFv) and is used to recognize and bind to tumor antigens.

3. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 1, characterized in that: The nucleotide sequence of the continuously truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.

1.

4. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 1, characterized in that: The chimeric antigen receptor structure also comprises an exon2 functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the exon2 functional region fragment is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3ζ signal domain.

5. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 4, characterized in that: The hinge region connects the scFv and the transmembrane domain, providing the flexibility required for CAR expression and antigen binding.

6. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 4, characterized in that: The nucleotide sequence of the exon2 functional region fragment is shown in SEQ ID NO.

1.

7. A chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 4, characterized in that: The novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

8. A method for preparing a chimeric antigen receptor structure for enhancing CAR-T cell killing activity as claimed in claim 1, characterized in that: It includes the steps of gene cloning, T cell collection and activation, gene transduction, in vitro amplification and screening, and preparation and reinfusion.

9. The method for preparing a chimeric antigen receptor structure for enhancing the killing activity of CAR-T cells according to claim 8, characterized in that: The gene transduction step utilizes a viral vector to transduce gene fragments encoding various domains of CAR into activated T cells.

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