阅读说明：本技术 一种基于FcγRⅢa的嵌合基因及其用途 (Fc gamma RIIa-based chimeric gene and application thereof ) 是由 孙振华 于 2016-03-18 设计创作，主要内容包括：本发明涉及一种基于FcγRⅢa的嵌合基因及其用途,所述嵌合基因包括依次串联的FcγRⅢa信号肽、FcγRⅢa细胞外区域、CD8α跨膜区域和胞内信号传导结构域,所述FcγRⅢa细胞外区域直接与CD8α跨膜区域连接；本发明通过将FcγRⅢa细胞外结构域直接与CD8α跨膜区连接,删除了常规嵌合抗原受体(CAR)分子设计中的CD8α铰合区,使得该种FcγRⅢa-CAR分子更有利于激活效应细胞,显著提高了FcγRⅢa-CAR分子对肿瘤细胞的杀伤能力；该种设计的FcγRⅢa-CAR分子与单克隆抗体药物联合可通用于多种肿瘤的细胞治疗。(The invention relates to a chimeric gene based on Fc gamma RIIa and application thereof, the chimeric gene comprises Fc gamma RIIa signal peptide, Fc gamma RIIa extracellular region, CD8 alpha transmembrane region and intracellular signal conduction domain which are sequentially connected in series, and the Fc gamma RIIa extracellular region is directly connected with the CD8 alpha transmembrane region; according to the invention, the Fc gamma RIIa extracellular domain is directly connected with the CD8 alpha transmembrane region, and a CD8 alpha hinge region in the conventional Chimeric Antigen Receptor (CAR) molecular design is deleted, so that the Fc gamma RIIa-CAR molecule is more beneficial to activating effector cells, and the killing capacity of the Fc gamma RIIa-CAR molecule on tumor cells is obviously improved; the designed Fc gamma RIIa-CAR molecule and the monoclonal antibody drug are combined to be universally used for cell therapy of various tumors.)

1. An Fc γ rliiia-based chimeric gene, comprising in series, in order, an Fc γ rliiia signal peptide, an Fc γ rliiia extracellular region, a CD8 α transmembrane region, and an intracellular signaling domain, wherein the Fc γ rliiia extracellular region is directly linked to a CD8 α transmembrane region.

2. The chimeric gene according to claim 1, wherein the Fc γ RIIa signal peptide has an amino acid sequence shown in SEQ ID NO. 8, and the coding gene sequence thereof is shown in SEQ ID NO. 3;

preferably, the Fc gamma RIIa extracellular region has an amino acid sequence shown as SEQ ID NO. 9, and a coding gene sequence thereof is shown as SEQ ID NO. 4;

preferably, the CD8 alpha transmembrane region has an amino acid sequence shown as SEQ ID NO. 10, and the coding gene sequence is shown as SEQ ID NO. 5.

3. The chimeric gene according to claim 1 or 2, characterized in that it further comprises a Kozak sequence; the Kozak sequence is shown as SEQ ID NO. 2;

preferably, the intracellular signaling domain is spliced by a costimulatory molecule and a cell activation signal;

preferably, the co-stimulatory molecule is any one or a combination of at least two of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 or CD83, preferably any one or a combination of two of CD28 or 4-1BB, more preferably 4-1 BB;

preferably, the 4-1BB has an amino acid sequence shown as SEQ ID NO. 11, and a coding gene sequence thereof is shown as SEQ ID NO. 6;

preferably, the cell activation signal is a CD3 zeta signaling domain;

preferably, the CD3 zeta signaling domain has the amino acid sequence as shown in SEQ ID No. 12 and its coding gene sequence is shown in SEQ ID No. 7.

4. The chimeric gene according to any one of claims 1 to 3, wherein said chimeric gene is prepared by sequential tandem splicing of a Kozak sequence, an FcyRIIa signal peptide, an FcyRIIIIa extracellular domain linked directly to a CD8 a transmembrane domain and free of a CD8 a hinge region, a CD8 a transmembrane domain, a costimulatory molecule, and a CD3 zeta signaling domain;

preferably, the chimeric gene has a nucleotide sequence shown as SEQ ID NO. 1.

5. A recombinant expression vector comprising the chimeric gene of any one of claims 1 to 4.

6. The chimeric gene of any one of claims 1-4 integrated in a manner comprising: retrovirus, adenovirus, lentivirus, herpes simplex virus, adeno-associated virus, vaccinia virus, baculovirus, lipofection, direct injection, sleeping beauty transposon system, mRNA transfection, mRNA electrotransfer, etc., preferably lentivirus and adeno-associated virus.

7. A cell expressing the chimeric gene of any one of claims 1 to 4 or comprising the recombinant expression vector of claim 5;

preferably, the cell is any one of a T cell, an NK cell or a DC cell; preferably, the first and second electrodes are formed of a metal,

the T cell is any one of a central memory T cell, an effector memory T cell or an effector T cell;

preferably, the NK cells are primary cultured NK cells or NK92 cell line.

8. Use of the cell according to claim 7 for the preparation of a medicament for the prophylactic and/or therapeutic and/or adjuvant treatment of malignant tumors or viral infectious diseases.

9. The use according to claim 8, wherein the cell is used in combination with a monoclonal antibody;

preferably, the monoclonal antibody is any one or a mixture of at least two of CD20, CD52, Her-1/2, EGFR, VEGF, CD117 or PD-1.

Technical Field

The invention relates to the technical field of tumor biotherapy, in particular to an Fc gamma RIIa-based chimeric gene and application thereof, and also relates to an Fc gamma RIIa-based genetically engineered immune cell and application thereof.

Background

Monoclonal antibodies have gradually become the mainstay of cancer therapy. The mechanism by which monoclonal antibodies exert therapeutic effects is primarily the killing of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In clinical applications of monoclonal antibodies, undesirable therapeutic effects are often observed. The reason is that effector cells of the ADCC effect of the monoclonal antibody medicine are exhausted after the patient is subjected to radiotherapy and chemotherapy, so that the monoclonal antibody medicine cannot fully exert the effect.

T cells (CART-19 cells) of the Chimeric Antigen Receptor (CAR) CD19 have been significantly successful in the treatment of CD 19-expressed B cell malignancies (Kochenderfer et al, 2010; Porter et al, 2011). Conventional CAR molecules were designed using the scFv of murine mab to bind CD3 ζ in combination with costimulatory molecules (CD28, 4-1BB, etc.). The Tumor Associated Antigen (TAA) aiming at different tissues needs to design scFv with corresponding specificity, and the constructed CAR molecule is only limited to aim at the tumor and has no universality, so that the clinical application of CAR technology is limited.

The design of conventional Chimeric Antigen Receptor (CAR) molecules mainly includes a CD8 α leader, a single chain variable region (scFv) formed by linking VH and VL with a Linker sequence, a CD8 α hinge region, a CD8 α transmembrane region, and an intracellular signaling region. The CD8 alpha hinge region provides flexible space for scFv to bind with antigen, and solves the problem of steric hindrance of scFv binding with antigen.

Fc γ rliiia (CD16a) is the only Fc receptor expressed on NK cells that can bind IgG to mediate ADCC. Fc γ riiia is a transmembrane glycoprotein containing a signal peptide sequence, an extracellular domain, a transmembrane region, and an intracellular domain. Wherein binding of the extracellular domain to the Fc portion of IgG mediates ADCC. Effector cells positively expressing Fc γ rliiia are key factors for exerting the ADCC effect of monoclonal antibodies. Clinically, the supplementation of effector cells positively expressing Fc gamma RIIa is urgently needed to improve the clinical curative effect of monoclonal antibody medicaments.

Therefore, how to develop an Fc γ rliiia-based chimeric gene and genetically engineered immune cells to solve the problems of the existing monoclonal antibody and chimeric antigen receptor technologies has become the focus of research.

Disclosure of Invention

Based on the principle that the Fc gamma RIIa is combined with an antibody Fc segment to generate ADCC effect, the invention designs the CAR molecule with the acting site of Fc gamma RIIa on the basis of optimizing the structure of the CAR molecule, and the CAR molecule not only can be used in combination with various different monoclonal antibody medicines and used for treating various tumors, but also can play the efficient killing function of the CAR molecule on tumor cells.

The inventors of the present invention have made extensive and intensive studies to achieve the above object, and as a result, have found that by directly linking the Fc γ rliia extracellular domain in the chimeric gene to the CD8 α transmembrane region, a CD8 α hinge region is not required, and the design of the Fc γ rliia-CAR molecule is more advantageous for activating effector cells, and can significantly improve the killing ability of the Fc γ rliiia-CAR molecule against various tumor cells, thereby achieving the above object.

Namely, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an Fc γ riia-based chimeric gene comprising, in series, an Fc γ riia signal peptide, an Fc γ riia extracellular region, a CD8 α transmembrane region and an intracellular signaling domain, the Fc γ riia extracellular region being directly linked to a CD8 α transmembrane region.

The present invention is different from the design of conventional CAR molecules. In the chimeric genes described in the present invention, the Fc γ riiia extracellular region is directly linked to the CD8 α transmembrane region, which deletes the CD8 α hinge region in the conventional CAR molecule. The design does not have the steric hindrance problem of antigen binding of a single chain variable region (scFv) formed by connecting a Linker sequence with VH and VL, and the inventor surprisingly finds that compared with a structure without deleting a CD8 alpha hinge region, the design of the Fc gamma RIIa-CAR molecule is more beneficial to activating effector cells and can significantly improve the killing capacity of the Fc gamma RIIIIa-CAR molecule on tumor cells.

The Fc gamma RIIa-based CAR molecule provided by the invention can not only identify tumor cells through monoclonal antibody drug targeting mediated effector cells, improve the clinical curative effect of monoclonal antibody drugs, but also play a role in killing the tumor cells of the CAR molecule; the CAR molecule and the monoclonal antibody drug which are designed by adopting the design can be universally used for cell therapy of various tumors.

According to the invention, the Fc gamma RIIa signal peptide has an amino acid sequence shown as SEQ ID NO. 8, and a coding gene sequence thereof is shown as SEQ ID NO. 3.

Compared with the conventional CD8 alpha leader sequence, the Fc gamma RIIa signal peptide adopted by the invention has the advantages that: the Fc gamma RIIa signal peptide is a part of the original structure of the Fc gamma RIIa gene, and is more favorable for guiding the protein of the Fc gamma RIIa extracellular region to penetrate a membrane and be cut at the later stage compared with a CD8 alpha leader sequence. Compared with an Fc gamma RIIa-CAR (defined as CD8 alpha leader-Fc gamma RIIIIa-CAR) designed by the invention aiming at a tumor cell killing capability test, the Fc gamma RIIa signal peptide Fc gamma RIIa-CAR (defined as Fc gamma RIIa signal peptide-Fc gamma RIIIIa-CAR) provided by the invention has the advantage that the selection of the Fc gamma RIIa signal peptide is obviously better than that of a CD8 alpha leader sequence designed by a conventional CAR molecule by constructing the Fc gamma RIIa-CAR (defined as CD8 alpha leader-Fc gamma RIIa-CAR) containing a CD8 alpha leader sequence.

According to the invention, the Fc gamma RIIa extracellular region has an amino acid sequence shown as SEQ ID NO. 9, and the coding gene sequence is shown as SEQ ID NO. 4.

According to the invention, the CD8 alpha transmembrane region has an amino acid sequence shown as SEQ ID NO. 10, and the coding gene sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 5.

According to the invention, the chimeric gene also contains a Kozak sequence; the Kozak sequence is shown in SEQ ID NO. 2.

The addition of a Kozak sequence before the signal peptide region is adopted in the invention, and the advantages are mainly reflected in that: the addition of a Kozak sequence can be used to enhance the translation efficiency of CAR molecules designed by the invention in eukaryotic cells (e.g., human T cells and NK cells).

The Kozak sequence, Fc gamma RIIa signal peptide and an extracellular region are sequentially spliced to form a functional region sequence combined with a monoclonal antibody Fc segment.

According to the invention, the intracellular signaling domain is formed by splicing a costimulatory molecule and a cell activation signal.

According to the invention, the co-stimulatory molecule is any one or a combination of at least two of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 or CD83, preferably any one or a combination of two of CD28 or 4-1BB, and more preferably 4-1 BB.

The invention adopts 4-1BB as a preferred costimulatory molecule, which has the advantages that: 4-1BB as a costimulatory molecule is more favorable for T cell survival in vivo than CD 28. According to the invention, the 4-1BB has an amino acid sequence shown as SEQ ID NO. 11, and a coding gene sequence thereof is shown as SEQ ID NO. 6. According to the invention, the cell activation signal

Is the CD3 zeta signaling domain.

According to the invention, the CD3 zeta signaling structural domain has an amino acid sequence shown as SEQ ID NO. 12, and a coding gene sequence is shown as SEQ ID NO. 7.

Preferably, the chimeric gene of the invention is formed by sequentially and serially splicing a Kozak sequence, an Fc gamma RIIa signal peptide, an Fc gamma RIIa extracellular region, a CD8 alpha transmembrane region, a costimulatory molecule and a CD3 zeta signaling domain, wherein the Fc gamma RIIa extracellular region is directly connected with the CD8 alpha transmembrane region and does not contain a CD8 alpha hinge region.

The Fc gamma RIIIIa-CAR molecular structure without a CD8 alpha hinge region is specifically as follows:

Kozak-Fc gamma RIIa signal peptide (signal peptide) -Fc gamma RIIa extracellular region (extracellular) -CD8 alpha transmembrane region (transmembrane region) -4-1BB-CD3 zeta. Preferred production of the invention

With the tandem mosaic structure, as a whole, the advantages that it has are mainly reflected in: kozak sequence can enhance translation efficiency in eukaryotic cells; the Fc gamma RIIa signal peptide is more favorable for guiding the protein in the extracellular region of the Fc gamma RIIa to penetrate a membrane and be cut at the later stage; the extracellular region of the Fc gamma RIIa is directly connected with the CD8 alpha transmembrane region, which is more favorable for activating the CAR molecule modified T cell; 4-1BB as a costimulatory molecule is more favorable for T cell survival in vivo. The CAR molecule formed by the series splicing structure can kill tumor cells in a targeted and efficient manner.

According to the invention, the chimeric gene preferably has the nucleotide sequence shown in SEQ ID NO 1. The nucleotide sequence and amino acid sequence of each of the above molecules can be obtained by a gene recombination method known in the field of molecular biology, for example, by using cDNA of a cell expressing the gene as a library and amplifying the gene by PCR, and preferably, the nucleic acid sequence is produced synthetically, rather than by cloning. The invention provides a universal preparation method of genetically engineered immune cells, which can not only mediate the recognition of effector cells to tumor cells through monoclonal antibody drugs, but also play the role of killing the tumor cells of CAR, and solve the problems of the existing monoclonal antibody and chimeric antigen receptor technologies.

The invention provides a preparation method of a genetically engineered immune cell based on Fc gamma RIIa, and a chimeric gene comprises a Kozak sequence, a signal peptide and an extracellular region of human Fc gamma RIIa, a CD8 alpha transmembrane domain, an intracellular costimulatory signaling region (4-1BB) and a CD3 zeta signaling domain. Wherein the extracellular region of Fc γ rliiia can bind to the Fc fragment of a variety of monoclonal antibodies, targetedly recognize the associated tumor, initiate ADCC and cytotoxicity of the CAR molecule.

To compare the design advantages of the present invention with those of the prior art, the chimeric gene of the present invention without the CD8 α hinge region was named Fc γ RIIa-BB-zeta, and the chimeric gene of the prior art containing the CD8 α hinge region was named Fc γ RIIIIa-CD 8 α -BB-zeta. The inventor finds that the CAR molecule without structural design of a CD8 alpha hinge region and a CD8 alpha transmembrane region are directly connected with an extracellular region of Fc gamma RIIa, so that the CAR molecule modified T cells are more favorably activated.

In a second aspect, the present invention also provides a recombinant expression vector comprising a chimeric gene as described in the first aspect.

In a third aspect, the invention also provides a cell expressing a chimeric gene as described in the first aspect or comprising a recombinant expression vector as described in the second aspect.

According to the present invention, the cell is any one of a T cell, an NK cell or a DC cell. Superior food

Optionally, the T cell is any one of a central memory T cell, an effector memory T cell or an effector T cell.

Preferably, the NK cells are primary cultured NK cells or NK92 cell line. The chimeric gene of the invention is introduced into effector cells and is expressed continuously. Methods of gene introduction are known in the art and specifically include physical, chemical and biological methods. The physical method comprises calcium phosphate transfection, microinjection, electroporation and the like, the chemical method comprises a liposome transfection system and the like, the biological method is mainly completed by constructing a viral vector, preferably a biological method, wherein the viral vector comprises adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus and the like, and preferably lentivirus.

The lentivirus vector of the chimeric gene of the invention is constructed by a method known in the field, and is cotransfected with a helper plasmid into 293T cells to obtain the lentivirus with the infection capacity and containing the chimeric gene of the invention.

Effector cells are defined herein as cells that are capable of integrating and transfecting the chimeric gene of the invention, mediating ADCC, and killing target cells. The effector cells may be primary cultured effector cells or effector cells derived from a cell line.

Sources of T cells include, but are not limited to, peripheral blood, bone marrow, lymph node tissue, cord blood, ascites, pleural effusion, preferably a source of peripheral blood. Enrichment of PBMCs was performed by Ficoll separation as known in the art, and CD3+ T cells were then isolated therefrom by flow sorting or MACS magnetic bead sorting for subsequent genetic modification.

Sources of NK cells include, but are not limited to, peripheral blood, bone marrow, lymph node tissue, cord blood, ascites, pleural effusion, and may also be derived from the NK92 cell line. Enrichment of PBMC was performed by Ficoll separation as known in the art, and CD3-CD16/56+ NK cells were isolated therefrom by flow sorting or MACS magnetic bead sorting for subsequent genetic modification.

Monoclonal antibodies for use in conjunction with effector cells include, but are not limited to, CD20, CD52, Her-1/2, EGFR, VEGF, CD117, or PD-1, and the like.

In a fourth aspect, the invention also provides the use of the cell according to the third aspect in the preparation of a medicament for the prevention and/or treatment and/or adjuvant treatment of malignant tumors or viral infectious diseases.

According to the present invention, the malignant tumor includes, but is not limited to, any one of lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, or prostate cancer.

In the present invention, the cells are used in combination with monoclonal antibodies.

Preferably, the monoclonal antibody is any one or a mixture of at least two of CD20, CD52, Her-1/2, EGFR, VEGF, CD117 or PD-1.

The immune cell of the chimeric Fc gamma RIIa gene designed by the invention is transfused into a patient body after being amplified in vitro, functional ADCC effector cells are supplemented, tumor cells are specifically identified under the mediation of a monoclonal antibody drug, and the CAR molecule plays a role in killing the tumor cells.

The clinical application of the monoclonal antibody needs a large amount of antibodies, but the therapeutic monoclonal antibody can be combined with effector cells in advance, namely, the therapeutic monoclonal antibody is frozen after cells are incubated by the corresponding therapeutic antibody in advance before T or NK cells are frozen and thawed and then directly returned for use, the use titer of the antibody is improved, the maximum value of ADCC effect is 0.1 mu g/ml of the therapeutic antibody, and the use amount of the clinical antibody is greatly reduced.

The engineered immune cell containing the chimeric gene can be combined with a therapeutic antibody, so that the clinical treatment effect of the monoclonal antibody is improved. Meanwhile, the immune cell modified by the chimeric gene can be expanded in vivo in the presence of the antibody, so that the tumor cell can be killed controllably by controlling the dosage of the injected antibody, and cytokine storm caused by conventional CAR molecule treatment is avoided.

Although the CART cell shows safety and effectiveness in clinical treatment, the CART cell has a wide application range at present, and only shows a good treatment effect in hematological tumors, but the CAR molecule can be used for treating various tumors, can also play a role in efficiently killing various tumor cells by the CAR molecule, and overcomes the limitation problem of tumor specific antigens when the CART is used for treating various tumors.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) the invention designs the CAR molecule with the acting site of Fc gamma RIIa on the basis of optimizing the CAR molecular structure, directly connects the Fc gamma RIIa extracellular region with the CD8 alpha transmembrane region, deletes the unique design of the CD8 alpha hinge region of the conventional CAR molecule, can be used together with various different monoclonal antibody medicaments for treating various tumors, is more favorable for activating effector cells compared with the structure containing the CD8 alpha hinge region, can further exert the high-efficiency killing function of the CAR molecule on tumor cells, and has the cell killing rate of more than 90 percent when the effective target ratio is 5: 1.

(2) The immune cell of the chimeric Fc gamma RIIa gene designed by the invention is transfused into a patient body after being amplified in vitro, functional ADCC effector cells are supplemented, tumor cells are specifically identified under the mediation of a monoclonal antibody drug, and the CAR molecule plays a role in killing the tumor cells.

(3) The invention can combine the therapeutic monoclonal antibody and the effector cell in advance, namely, the therapeutic monoclonal antibody is frozen before the T or NK cell is frozen and the cell is incubated with the corresponding therapeutic antibody in advance and then is frozen and thawed and then directly returned for use, thereby improving the use titer of the antibody and greatly reducing the dosage of clinical antibody; in addition, the cell survival rate can still reach more than 90 percent after the cryopreservation recovery.

(4) The immune cell of the chimeric Fc gamma RIIa gene designed by the invention can be expanded in vivo in the presence of an antibody, and the amount of the injected antibody is controlled, so that tumor cells can be killed controllably, and cytokine storm caused by conventional CAR molecule treatment is avoided. Drawings

FIG. 1 is a schematic structural view of a chimeric gene of the present invention.

FIG. 2 shows the target gene portion released by double digestion of the chimeric gene Fc γ RIIa-BB-zeta with BamH1 and Sal1 in the lentiviral expression vector pLVX-EF 1. alpha. with 1269bp target gene fragment, Lane 1 showing the standard nucleic acid molecular weight, and Lane 2 showing the product of double digestion with BamH1 and Sal 1.

FIG. 3 is a graph showing the results of detection of expression of endogenous CD3 and fusion gene Fc γ RIIa-BB-zeta in T cells of chimeric Fc γ RIIIIa-BB-zeta gene by Western Blotting, lane 1 is untransfected T cells as a negative control, lane 2 is 5MOI lentivirus transfected T cells, and lane 3 is 10MOI lentivirus transfected T cells.

FIG. 4 is a graph showing the results of virus transduction efficiency using a flow cytometer; wherein, FIG. 4-A is a CD3PE and CD16FITC double-labeled scattergram, which indicates that the cultured cells are CD3 positive, 83.57% of CD3 positive cells are CD16 positive, indicating that the virus transduction efficiency is 83.57%; FIG. 4-B is a straight-peak CD16FITC chart showing transduction efficiency in total cells.

FIG. 5 is a graph showing the results of measuring the phosphorylation level of CD3 ζ by flow cytometry, wherein FIG. 5-A is the phosphorylation level of Fc γ RIIa-CD 8 α -BB- ζ, and FIG. 5-B is the phosphorylation level of Fc γ RIIIIa-BB- ζ.

FIG. 6 is a graph showing the test of the killing ability of the chimeric gene Fc γ RIIa-BB-zeta of the present invention against target cells, wherein FIG. 6A is a graph showing the test of the killing ability of the chimeric gene Fc γ RIIIIa-BB-zeta against target cells Raji; FIG. 6-B is a graph showing the killing ability of the chimeric gene Fc γ RIIa-BB-zeta against target cell SKOV 3; FIG. 6-C is a graph showing the killing ability of the chimeric gene Fc γ RIIa-BB-zeta against the target cell ANT 1; FIG. 6-D is a graph showing the killing ability of the chimeric genes Fc γ RIIa-BB-zeta and CD8 α leader-Fc γ RIIIIa-CAR on target cells.

FIG. 7 is a graph showing the detection of IFN-. gamma.secretion levels of Raji, SKOV3 and ANT1 by the chimeric gene Fc.gammaRIIa-BB-zeta of the present invention.

FIG. 8 is a diagram showing the killing ability of NK92 cells of the chimeric Fc γ RIIIIa-BB-zeta gene of the present invention.

FIG. 9 is a graph showing a comparison of cell viability of Fc γ RIIIIa-BB- ζ -NK cells prepared according to the present invention before and after cryopreservation.

FIG. 10 is a graph showing a comparison of the cell killing ability of Fc γ RIIIIa-BB- ζ -NK cells prepared according to the present invention before cryopreservation and after cryopreservation recovery.

The present invention is described in further detail below. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. To better illustrate the invention and to facilitate the understanding of the technical solutions thereof, typical but non-limiting examples of the invention are as follows:

fig. 1 shows the structure of the chimeric gene of the invention, and the molecular structure of the Fc γ riiia-CAR is specifically as follows:

Kozak-Fc γ RIIa Signal peptide-Fc γ RIIIIa extracellular region-CD 8 α transmembrane region-4-1 BB-CD3 ζ

The chimeric gene of the invention does not contain a CD8 alpha hinge region and is formed by directly connecting an Fc gamma RIIa extracellular region with a CD8 alpha transmembrane region.