WO2022198611A1 - Car chimeric antigen receptor sequence and car-nk cells applying same - Google Patents

Abstract

The present invention provides a CAR chimeric antigen receptor sequence, comprising a single-chain antibody structure, an extracellular domain structure, a transmembrane domain structure, and an intracellular domain structure. A protein polypeptide sequence of the extracellular domain structure is selected from fragments of a CD8α protein polypeptide sequence. A protein polypeptide sequence of the transmembrane domain structure is selected from fragments of an NKG2D protein polypeptide sequence. The intracellular domain structure comprises a 2B4 intracellular domain structure or/and a CD3ζ intracellular domain structure. A protein polypeptide sequence of the 2B4 intracellular domain structure is selected from fragments of a 2B4 protein polypeptide sequence. A protein polypeptide sequence of the CD3ζ intracellular domain structure is selected from fragments of a CD3ζ protein polypeptide sequence. The CAR chimeric antigen receptor sequence provided by the present invention can effectively activate immune functional cells, and can enable the immune functional cells embedded with the CAR chimeric antigen receptor sequence to efficiently recognize malignant tumor cells.

Description

A CAR chimeric antigen receptor sequence and CAR-NK cells using the same technical field

The invention belongs to the field of cellular immunotherapy, and in particular, relates to a CAR chimeric antigen receptor sequence and a CAR-NK cell using the same.

Background technique

Malignant tumor is one of the major diseases that endanger human health. Traditional tumor treatment methods such as surgery, radiotherapy, and chemotherapy have been the main strategies for tumor treatment in recent decades. However, patients will develop resistance to drugs and radiotherapy, resulting in high frequency of tumor recurrence. Tumor cell therapy has attracted great attention due to its advantages of targeting, remarkable effect, and few side effects, and has gradually become an important means of comprehensive tumor therapy. Hot spots and development directions of research and clinical applications.

Natural killer cells (NK cells) do not need pre-stimulation to exert their killing activity, and have great potential in the treatment of human malignant tumors based on cell therapy. NK cells can spontaneously lyse tumor cells in the absence of T and B cells. NK cells share many features with cytotoxic T cells, including common precursor cells, rearrangement of cell surface receptor molecules, and granzyme-dependent killing mechanisms. However, NK cells are also unique in that they do not require pre-stimulation to kill and are not MHC-restricted in killing tumor cells. This makes NK cells widely used in adoptive tumor cell immunotherapy, which refers to the return of exogenously engineered cells to patients for clinical applications.

NK cells isolated and expanded in vitro can be used for autologous or allogeneic therapy. However, the method of depleting T cells from leukocytes in a single donor's blood to obtain NK cells yields unstable NK cell yields, and the numbers are not sufficient for reinfusion. In addition, NK cells are very difficult to culture in vitro, far from meeting the requirements of cellular immunotherapy. Moreover, many NK cells in tumor patients can no longer function normally. Even in vitro, many activators (such as IL-2, IL-12, IL-5, IL-18, etc.) need to be added to enhance the response of NK cells to tumor cells.

Although there are many methods to prepare NK cells at present, the low differentiation efficiency of NK cells makes the price of NK cell drugs high, and the killing activity is difficult to control, which is not conducive to large-scale production and application. Based on this, the in vitro activation and expansion of NK cells has practical significance for the further promotion and application of NK cells in tumor cell immunotherapy.

Tumor chimeric antigen receptor (CAR) therapy refers to a new type of precise targeted therapy that uses chimeric antigen receptors to treat tumors on immune cells. In recent years, it has achieved good results in clinical tumor treatment through optimization and improvement. The effect is a very promising new tumor immunotherapy method that can be precise, fast, efficient, and has the potential to cure cancer. Usually, CAR activates immune cells (T cells, NK cells) through genetic engineering technology, and installs a positioning navigation device CAR, and reorganizes ordinary T cells and NK cells into CAR-T cells and CAR-NK cells correspondingly. CAR-T cells and CAR-NK cells can use their chimeric CARs to specifically identify tumor cells in the body, and release a large number of various effectors through immunization, which can efficiently kill tumor cells, so as to achieve the purpose of treating malignant tumors. . In addition, the immune cells that have been positioned and knocked into the CAR vector can be transplanted back into the original patient's body to avoid the attack of the autoimmune system.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a CAR chimeric antigen receptor sequence and a CAR-NK cell using the same, so as to effectively activate immune function cells and make immune function cells efficiently kill tumor cells.

According to one aspect of the present invention, a CAR chimeric antigen receptor sequence is provided, including a single-chain antibody structure, an extracellular domain structure, a transmembrane domain structure and an intracellular domain structure; the protein polypeptide sequence of the extracellular domain structure is selected from CD8α Fragments of protein polypeptide sequences; protein polypeptide sequences of transmembrane domain structures are selected from fragments of NKG2D protein polypeptide sequences; intracellular domain structures include 2B4 intracellular domain structures or/and CD3ζ intracellular domain structures, protein polypeptides of 2B4 intracellular domain structures The sequence is selected from the fragment of the 2B4 protein polypeptide sequence, and the protein polypeptide sequence of the CD3ζ intracellular domain structure is selected from the fragment of the CD3ζ protein polypeptide sequence.

Preferably, the intracellular domain structure includes the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure, and the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure are connected in series in sequence.

Preferably, the extracellular domain structure includes a first extracellular domain structure and a second extracellular domain structure; the first extracellular domain structure, the single-chain antibody structure, the second extracellular domain structure, the transmembrane domain structure, and the 2B4 intracellular domain The structure and CD3ζ intracellular domain structure are connected in series; the single-chain antibody structure is the anti-mesothelin scFv encoded by SEQ ID NO.7.

Preferably, its protein polypeptide sequence is SEQ ID NO.10.

Preferably, the extracellular domain structure, the single-chain antibody structure, the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure are connected in series in sequence; the single-chain antibody structure is the anti-epidermal growth factor receptor encoded by SEQ ID NO.12. Body scFv.

Preferably, its protein polypeptide sequence is SEQ ID NO.14.

According to one aspect of the present invention, a CAR chimeric antigen receptor sequence is provided, the protein polypeptide sequence of which is not less than 80% similar to the protein sequence of the above-mentioned CAR chimeric antigen receptor series.

According to another aspect of the present invention, there is provided a CAR-NK cell whose AAVS1 site is chimeric with a sequence vector comprising the ER1a promoter and the above-mentioned CAR chimeric antigen receptor sequence; in the sequence vector, the CAR chimeric antigen receptor is The body sequence is located downstream of the ER1a promoter.

According to another aspect of the present invention, the application of CAR-NK cells in the preparation of anti-cancer drugs is provided.

Preferably, the anti-cancer drug is an anti-malignant mesothelioma drug.

The CAR chimeric antigen receptor sequence provided by the present invention can effectively activate immune function cells, and can make the chimeric immune function cells efficiently recognize malignant tumor cells. Optionally, the CAR chimeric antigen receptor sequence provided by the present invention adopts anti-mesothelin scFv or anti-epidermal growth factor receptor scFv as the single-chain antibody structure contained therein, so that the chimeric immune function cells can efficiently Identification of malignant mesothelioma cells.

The sequence vector including the above-mentioned CAR chimeric antigen receptor sequence is chimeric in the AAVS1 site of NK cells. The location of the knock-in is clear, and it will not interfere with other genes of the host cell, and will not cause malignant transformation of the host cell due to transgene. At the same time, the present invention sets the EF1a promoter that can be uniformly and highly expressed in various cells upstream of the above CAR chimeric antigen receptor sequence, so that CAR-NK cells with biological activity and functions can be successfully and efficiently obtained, and the stem cells can be The proportion of differentiation to NK cells was significantly increased, and the proportion of differentiation was greater than 90%.

Optionally, the sequence vector used in the present invention may include an IRES sequence, which can make mRNA translation and fusion with exogenous cDNA independent of the 5' cap structure. Optionally, the sequence vector used in the present invention may include a T2A sequence, so that the entire mRNA generated using the sequence vector can be self-spliced into two independent proteins. Optionally, the sequence vector used in the present invention may include a GFP sequence or a Puro sequence, so that the CAR-NK cells constructed by the present invention can be identified and detected by fluorescence analysis or drug screening accordingly.

In conclusion, the CAR-NK cells provided by the present invention have the advantages of stability, high efficiency and high safety, and are suitable for large-scale production and application.

Description of drawings

1 is a schematic diagram of the sequence vector constructed in Example 1 for targeting and knocking into the AAVS1 site of CAR-NK cells;

FIG. 2 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 before injection of CAR-NK cells I;

FIG. 3 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 after 1 hour of injection of CAR-NK cells I;

FIG. 4 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 after 3 hours of injection of CAR-NK cells I;

Figure 5 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 5 hours after injection of CAR-NK cells I;

Figure 6 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 7 hours after injection of CAR-NK cells I.

Detailed ways

In order for those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not All examples.

The term "chimerism" as used in the following examples refers to the biological process of obtaining specialized cells (eg, NK cells) by non-specialized CARs under controlled conditions when cultured in vitro. Differentiation is controlled by the interaction of cellular genes with extracellular physical and chemical conditions, usually via signaling pathways involving proteins embedded on the cell surface.

As used in the following examples, the term "about", unless explicitly stated or clear from context, is understood to mean within a range of normal tolerance in the art, eg, within 2 standard deviations of the mean. About 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value Inside.

Example 1

The CAR chimeric antigen receptor sequences (CAR sequences) involved in this example include CAR-A sequences, CAR-AA sequences and CAR-B sequences.

1. Construction of CAR-AA sequence

1.1 Design the nucleotide sequence encoding the CAR-A sequence

CAR-A sequence includes extracellular domain structure, single chain antibody structure, transmembrane domain structure and intracellular domain structure. The extracellular domain structure includes a first extracellular domain structure and a second extracellular domain structure: the nucleotide sequence encoding the first extracellular domain structure is SEQ ID NO.1, and SEQ ID NO.1 belongs to the nucleoside encoding CD8α protein Fragment of acid sequence (NCBI Reference Sequence: NM_171827); the nucleotide sequence encoding the second extracellular domain structure is SEQ ID NO.2, and SEQ ID NO.2 belongs to the nucleotide sequence encoding CD8α protein (NCBI Reference Sequence: NM_001768). The nucleotide sequence encoding the structure of the transmembrane region is SEQ ID NO.3, and SEQ ID NO.3 is a fragment of the nucleotide sequence encoding the NKG2D protein (NCBI Reference Sequence: NM_007360.3). The intracellular domain structure includes the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure: the nucleotide sequence encoding the 2B4 intracellular domain structure is SEQ ID NO.4, and SEQ ID NO.4 belongs to the nucleotide sequence encoding the 2B4 protein ( NCBI Reference Sequence: fragment of NM_001166663.1); the nucleotide sequence encoding CD3ζ intracellular domain structure is SEQ ID NO.5, and SEQ ID NO.5 belongs to the nucleotide sequence encoding CD3ζ protein (NCBI Reference Sequence: NM_198053. 2) fragment; SEQ ID NO.6 formed in series with SEQ ID NO.4 and SEQ ID NO.5 is the nucleotide sequence encoding the intracellular region structure of the CAR-A sequence. The single-chain antibody structure is anti-mesothelin scFv (anti-mesothelin scFv), and the nucleotide sequence encoding the anti-mesothelin scFv is SEQ ID NO.7.

In the CAR-A sequence, the first extracellular domain structure, the single-chain antibody structure, the second extracellular domain structure, the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure are connected in series in sequence, encoding the CAR-A sequence. The nucleotide sequence is SEQ ID NO.8.

1.2 Optimization of the nucleotide sequence encoding the CAR-A sequence

The nucleotide sequence SEQ ID NO.8 encoding the CAR-A sequence is optimized to obtain SEQ ID NO.9, and the protein polypeptide SEQ ID NO.10 obtained after translating SEQ ID NO.9 is the CAR-AA sequence.

2. Construction of CAR-B sequences

CAR-A sequence includes extracellular domain structure, single chain antibody structure, transmembrane domain structure and intracellular domain structure. The nucleotide sequence encoding the structure of the extracellular domain is SEQ ID NO. 11, and SEQ ID NO. 11 is a fragment of the nucleotide sequence encoding the CD8α protein (NCBI Reference Sequence: NM_171827). The nucleotide sequence encoding the structure of the transmembrane region is SEQ ID NO.3, and SEQ ID NO.3 is a fragment of the nucleotide sequence encoding the NKG2D protein (NCBI Reference Sequence: NM_007360.3). The intracellular domain structure includes the 2B4 intracellular domain structure and the CD3 ζ intracellular domain structure: the nucleotide sequence encoding the 2B4 intracellular domain structure is SEQ ID NO.4, and SEQ ID NO.4 belongs to the nucleotide sequence encoding the 2B4 protein (NCBI Reference Sequence: NM_001166663.1) fragment; the nucleotide sequence encoding the intracellular domain structure of CD3ζ is SEQ ID NO.5, and SEQ ID NO.5 belongs to the nucleotide sequence encoding CD3ζ protein (NCBI Reference Sequence: NM_198053 .2) fragment; SEQ ID NO.6 formed by concatenation of SEQ ID NO.4 and SEQ ID NO.5 is the nucleotide sequence encoding the intracellular region structure of the CAR-B sequence. The structure of the single chain antibody is anti-epidermal growth factor receptor scFv (anti-HER3 scFv), and the nucleotide sequence encoding the anti-epidermal growth factor receptor scFv is SEQ ID NO.12.

In the CAR-B sequence, the extracellular domain structure, single-chain antibody structure, transmembrane domain structure, 2B4 intracellular domain structure and CD3ζ intracellular domain structure are connected in series in sequence, and the nucleotide sequence encoding the CAR-B sequence is SEQ ID NO. 13. The protein polypeptide SEQ ID NO.14 obtained after translating SEQ ID NO.13 is the CAR-B sequence.

3. Construction of CAR sequence vector

The nucleotide sequences of CAR-AA sequence and CAR-B sequence were amplified by PCR technology, and then the CAR-AA sequence and CAR-B sequence were cloned into sequences containing T2A sequence, EF1a promoter sequence, GFP sequence, and Puro sequence, respectively. In the sequence vector with the IRES sequence, a sequence vector with the EF1a promoter sequence, the CAR sequence, the T2A sequence, the GFP sequence, the IRES sequence and the Puro sequence in series is obtained (as shown in Figure 1). Among them, the sequence vector using CAR-AA as its CAR sequence is marked as sequence vector I, and the sequence vector using CAR-B as its CAR sequence is marked as sequence vector II.

2. Construction of CAR-NK cells

Select endonuclease to linearize sequence vector I and sequence vector II, respectively, and then use the corresponding reagents of CRISPR/Cas9 to locate the linearized sequence vector I and linearized sequence vector II respectively into the AAVS1 site of NK cells. The NK recombinant cells chimeric with sequence vector I were labeled as CAR-NK cells I, and the NK recombinant cells chimeric with sequence vector II were labeled as CAR-NK cells II. The CAR-NK cells successfully chimeric with the above sequence vector can express green fluorescent protein and puromycin at the same time, and the fluorescent protein-positive cells can be screened by fluorescence detection, or the puromycin-positive cells can be screened by using puromycin antibody, thereby Screen the CAR-NK cells that successfully chimeric the above sequence vector.

Example 2

1. Experiment setup method

In this example, NK cells provided by the same volunteer were used to prepare CAR-NK cells I and CAR-NK cells II according to the method provided in Example 1. Two groups of treatment groups (including treatment I and treatment II) and one group of control groups were set up to carry out the following in vitro tumor killing effect experiments according to the following steps. Specifically, the following experimental operations were carried out with CAR-NK cells I for treatment I, and CAR for treatment II. - NK cells II were subjected to the following experimental procedures, and the control group was subjected to the following experimental procedures using NK cells from the same volunteer. Each treatment group and control were detected using the CCK-8 method (see: Human Leukocyte Antigen-G Inhibits the Anti-TumorEffect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 in Gastric Cancer, Rui Wan Zi-Wei Wang Hui Li, et al.) The killing effect of the test cells in the group on malignant mesothelioma cells in vitro:

(1) Malignant mesothelioma cells (40,000 cells) were evenly spread in 24 wells one day before the experiment, and waited for the cells to adhere to the wall;

(2) After the experiment started, NK cells, CAR-NK cells I, CAR-NK cells II and CAR-NK cells III were plated in 24 wells (40,000 cells) according to the ratio of effector to target of 1:1. The system is 1ml, incubated at 37 degrees for 7 hours;

(3) After 7 hours of action, discard the medium, add PBS to gently rinse twice, add 200ul of CCK8 reagent, and act at 37°C for 1-4 hours;

(4) The killing rate of tumor cells was calculated by reading the plate at 450 nm.

2. Experimental results

By comparing Figures 2-6, it can be seen that after the malignant mesothelioma cells were incubated with CAR-NK cells I for 7 hours, the malignant mesothelioma cells were significantly reduced, which proves that CAR-NK cells I treated with I can effectively eliminate malignant cells. Skin tumor cells.

The experimental results of the evaluation of the in vitro tumor killing effect of the test cells in the treatment group and the control group set in this example are shown in Table 1. Compared with the tumor cell killing rate corresponding to the control group, the tumor cells corresponding to the three treatment groups The kill rate is significantly higher.

Table 1 Killing rate of malignant mesothelioma cells

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent substitutions are made without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

A CAR chimeric antigen receptor sequence, characterized in that: Including single-chain antibody structure, extracellular domain structure, transmembrane domain structure and intracellular domain structure; The protein polypeptide sequence of the extracellular domain structure is selected from the fragment of the CD8α protein polypeptide sequence; The protein polypeptide sequence of the transmembrane region structure is selected from the fragment of the NKG2D protein polypeptide sequence; The intracellular domain structure includes the 2B4 intracellular domain structure or/and the CD3ζ intracellular domain structure, the protein polypeptide sequence of the 2B4 intracellular domain structure is selected from the fragment of the 2B4 protein polypeptide sequence, and the CD3ζ intracellular domain structure protein. The polypeptide sequence is selected from a fragment of the CD3ζ protein polypeptide sequence. The CAR chimeric antigen receptor sequence according to claim 1, characterized in that: The intracellular domain structure includes the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure, The transmembrane domain structure, the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure are connected in series in sequence. The CAR chimeric antigen receptor sequence according to claim 2, characterized in that: The extracellular domain structure includes a first extracellular domain structure and a second extracellular domain structure; The first extracellular domain structure, the single-chain antibody structure, the second extracellular domain structure, the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure are connected in series in sequence; The single-chain antibody structure is an anti-mesothelin scFv encoded by SEQ ID NO.7. The CAR chimeric antigen receptor sequence according to claim 3, characterized in that: its protein polypeptide sequence is SEQ ID NO.10. The CAR chimeric antigen receptor sequence according to claim 2, characterized in that: The extracellular domain structure, the single-chain antibody structure, the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3ζ intracellular domain structure are connected in series in sequence; The single-chain antibody structure is an anti-epidermal growth factor receptor scFv encoded by SEQ ID NO.12. The CAR chimeric antigen receptor sequence according to claim 5, characterized in that: its protein polypeptide sequence is SEQ ID NO.14. A CAR chimeric antigen receptor sequence, characterized in that its protein polypeptide sequence is not less than 80% similar to the protein sequence of the CAR chimeric antigen receptor series according to claim 4. A CAR chimeric antigen receptor sequence, characterized in that its protein polypeptide sequence is not less than 80% similar to the protein sequence of the CAR chimeric antigen receptor series according to claim 6. A CAR-NK cell, characterized by: Its AAVS1 site is chimeric with a sequence vector comprising the ER1a promoter and the CAR chimeric antigen receptor sequence according to any one of claims 1-8; In the sequence vector, the CAR chimeric antigen receptor sequence is located downstream of the ER1a promoter. The application of CAR-NK cells in the preparation of anti-cancer drugs according to claim 9.

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