CN110699371A

CN110699371A - Fc gamma RIIa-based chimeric gene and application thereof

Info

Publication number: CN110699371A
Application number: CN201910990200.9A
Authority: CN
Inventors: 孙振华
Original assignee: Jiangsu Pro Health Biotech Co Ltd
Current assignee: Jiangsu Pro Health Biotech Co Ltd
Priority date: 2016-03-18
Filing date: 2016-03-18
Publication date: 2020-01-17
Also published as: CN105647946B; CN105647946A

Abstract

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.

Description

Fc gamma RIIa-based chimeric gene and application thereof

The application is a divisional application, the application number of the original application is 201610156775.7, the application date is 2016, 3 and 18, and the name of the invention is 'an Fc gamma RIIa-based chimeric gene and application thereof'.

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.

Example 1 double restriction enzyme identification of the Fc γ RIIIIa-BB-zeta lentivirus expression vector by the restriction enzyme BamH/Sal1

FIG. 2 shows the target gene portion of the chimeric gene Fc γ RIIa-BB-zeta, 1269bp, which is released by double digestion with BamH1 and Sal1 on the lentiviral expression vector pLVX-EF1 α. Lane 1 shows the nucleic acid molecular weight standards, and Lane 2 shows the double cleavage products of BamH1 and Sal 1.

And (3) sending the plasmid with correct double enzyme digestion identification to Shanghai biological engineering Limited company to sequence the inserted chimeric gene fragment. The plasmid with the correct sequencing was named pLVX-Fc γ RIIa-BB-zeta.

Example 2 preparation of Fc γ RIIa-BB-zeta lentivirus

The Fc γ RIIa-BB-zeta chimeric gene was designed and constructed as described in the present invention. Lentiviral production and purification of the chimeric gene Fc γ RIIa-BB-zeta was carried out as follows:

1. extracting plasmids by adopting a Qiagen Endofree Plasmid Maxi Kit;

2. preparing 293T cells of a 100mm culture dish in advance;

3. digesting the cells by pancreatin, sucking 10ml of complete culture medium by an electric pipette, blowing all the cells into single cell suspension, and transferring the single cell suspension into other 100mm culture dishes according to a proportion; 37 ℃ and 5% CO₂The incubator stays overnight;

4. transfecting when the cell fusion degree is 70% -80%;

5. replacing the cells with a liquid, and replacing the whole amount of the cells with a fresh serum-free DMEM medium;

6. preparing a plasmid mixed solution: taking a new 1.5mL centrifuge tube, and adding 0.5mL of DMEM and plasmid;

7. preparing a PEI mixed solution: taking a new 1.5mL centrifuge tube, adding 0.5mL DMEM and 30 mul PEI, and mixing uniformly;

8. adding the PEI mixed solution into the plasmid mixed solution, uniformly mixing, and standing for 15min at room temperature;

9. taking a dish of cells, dropwise adding 1ml of mixed solution in a centrifugal tube, and distributing a transfer reagent into the whole culture dish as far as possible;

10. the Petri dish was returned to 5% CO₂Placing in an incubator for 6 h;

11. after transfection for 6h, the solution was changed to 10ml complete medium;

12. after transfection for 48h, collecting the same virus packaging supernatant together, and discarding the culture dish;

13. subpackaging in 50ml centrifuge tube, centrifuging at 500g room temperature for 10min, and removing cells and large debris;

14. filtering the supernatant with 0.22 μ M needle filter, and standing at 4 deg.C for purification;

15. using AKTA flux 6 machine, 5L virus 0.65 μm hollow fiber microfiltration column;

16. using AKTA flux 6 machine, virus 300kD hollow fiber ultrafiltration column;

17. concentrating the virus to 200 ml;

18. using AKTA pure 150Protein Purification System for virus Purification;

19. the virus was eluted with 50ml PBS;

20. the virus was filtered through a 0.22 μ M filter tip and the virus tubes were aliquoted. Lentiviral coating systems include, but are not limited to, three-plasmid expression systems and four-plasmid expression systems, with four-plasmid expression systems (plasmid of interest, pRRE, pREV, VSVG) being preferred in this example. Lentivirus purification systems include, but are not limited to, ultracentrifugation, dialysis, ultrafiltration, and the like, with hollow fiber ultrafiltration being preferred in the present invention.

Example 3 preparation of cell products of chimeric Fc γ riiia-BB-zeta genes cells were derived from patients or healthy donors and Peripheral Blood Mononuclear Cells (PBMCs) were obtained by intravenous blood sampling or apheresis. The T cell culture method employs a method of activating T cells by coating a culture flask with a CD3, CD28 monoclonal antibody, or a method of activating T cells by paramagnetic polystyrene beads coated with CD3 and CD28 monoclonal antibodies, and the NK cell culture method is performed as described (Hiroyuki et al, cancer Res 2009; 69: 4010-. Levoviral transduction was performed as described (Levine et al, 2006, Proc Natl Acadsi USA103: 17372-.

Example 4 detection of chimeric Gene Fc γ RIIa-BB-zeta modified T cell phenotypic characterization by flow cytometry

T cells were cultured by the method of example 3 to day 14, and expression of endogenous CD3 and fusion gene Fc γ RIIa-BB-zeta was detected in T cells of chimeric Fc γ RIIa-BB-zeta gene by Western Blotting. As shown in fig. 3.

FIG. 3 shows T cells transfected with the fusion gene Fc γ RIIa-BB-zeta lentivirus and examined for endogenous CD3 and fusion protein expression using Western Blotting. Lane 1 is untransfected T cells as a negative control, lane 2 is 5MOI lentivirus transfected T cells, lane 3 is 10MOI lentivirus transfected T cells, and the internal reference is β -actin. The results show successful expression of the fusion protein.

Example 5 detection of viral transduction efficiency

10MOI fusion gene Fc gamma RIIa-BB-zeta lentivirus infects T cells for 5 days, samples are taken and flow cytometry is carried out to detect CD3PE and CD16FITC expression, and the expression level of CD16 represents the transduction efficiency of the lentivirus.

As shown in FIGS. 4-A and 4-B, the expression level of CD16 was 83.57%, i.e., the transduction efficiency of the fusion gene Fc γ RIIIIa-BB-zeta lentivirus was 83.57%.

Example 6 flow cytometry detection of phosphorylation level of CD3 ζ

T cells were infected with 10MOI fusion gene Fc γ RIIa-BB-zeta lentivirus using PBMC from the same donor and cultured until day 5. The control group was cultured to day 5 by infecting T cells with 10MOI fusion gene Fc γ RIIa-CD 8 α -BB-zeta lentivirus. Taking Raji cells positive to CD20 1X 10⁵1. mu.g/ml Rituximab antibody was added to the transfected T cells cultured to day 5 at 1X 10⁶The cells were incubated for 2 hours. The phosphorylation level of CD3 ζ was detected by flow cytometry. The results are shown in FIG. 5.

As shown in FIG. 5-A, the phosphorylation level of Fc γ RIIII a-CD8 α -BB-zeta was 50.45%, and as shown in FIG. 5-B, the phosphorylation level of Fc γ RIIII a-BB-zeta was 79.77%, which were significantly different. The CAR molecular structure Fc gamma RIIa-BB-zeta designed by the invention is more beneficial to phosphorylation of CD3 zeta and activation of transfected T cells compared with the prior art Fc gamma RIIIIa-CD 8 alpha-BB-zeta, and indicates higher killing capacity on tumor cells.

Example 7 cell killing assay

Antibody incubation was performed at a concentration of 0.1 μ g/ml, and killing assay was performed on Raji cells (CD20+, rituximab), SKOV3 cells (Her2+, trastuzumab), ANT1 cells (CCR4+, mogamulizumab) at E: T ═ 5:1, 2.5:1, 1.25:1, 0.6:1, 0.3:1, 5 target-effect ratios.

As shown in FIGS. 6-A, 6-B and 6-C, the killing ability of the Fc gamma RIIa-BB-zeta of the invention to target cells was significantly better than that of Fc gamma RIIIIa-CD 8 alpha-BB-zeta under the condition of co-incubation of corresponding antibodies at 0.1 mu g/ml for three cells of Raji, SKOV3 and ANT1 under different effective-to-target ratios.

Figure 6-D shows that Fc γ riiia signal peptide-Fc γ riiia-CAR kills target cells significantly better than CD8 α leader-Fc γ riiia-CAR with rituximab antibody 0.1 μ g/ml co-incubation for Raji cells at different effective target ratios. The Fc γ rliiia signal peptide is preferred as the signal peptide in the present invention without the conventional CD8 α leader sequence.

Example 8 measurement of IFN-. gamma.secretion amount

The level of IFN- γ secretion was measured using BD CBA assay kit after incubation of Raji cells (CD20+, rituximab), SKOV3 cells (Her2+, trastuzumab), ANT1 cells (CCR4+, mogamulizumab) for 4 hours at a concentration of 0.1. mu.g/ml for E: T1: 1. The results of the control group incubated with K562 and the corresponding antibody are shown in FIG. 7.

The results show that the secretion level of cytokine IFN-gamma is significantly higher than that of Fc gamma RIII a-CD8 alpha-BB-zeta group in the case of co-incubation of 0.1. mu.g/ml of corresponding antibody for three cells of Raji, SKOV3 and ANT 1.

Example 9 Fc Gamma RIIa-BB-zeta lentivirus infection NK92 assay

NK92 was not expressed by CD16a itself, and the specific killing ability of Fc γ RIIIIa-BB-zeta was verified by transducing the NK92 cell line with Fc γ RIIIIa-BB-zeta, using untransfected NK92 as control. Where NK92 has no ADCC effect, this experiment was used to directly demonstrate the advantages of the CAR molecule design.

NK92 cells were cultured with reference to ATCC instructions and 10MOI Fc γ R IIIa-BB-zeta lentivirus infected cultured NK92 cells were investigated for killing of Raji cells (CD20+, rituximab), SKOV3 cells (Her2+, trastuzumab), ANT1 cells (CCR4+, mogamulizumab) by co-incubation with untransfected NK92 cells as control and antibodies. NK92 cells themselves lacked Fc γ rliiia expression, the results of which are shown in fig. 8.

The control experiment proves that the NK92 cells of the chimeric Fc gamma RIIIIa-BB-zeta gene have ADCC killing capability; the results also show that the killing ability of NK92 cells expressing the chimeric gene Fc gamma RIIIIa-BB-zeta is significantly improved compared to untransfected NK92 cells, indicating that NK92 cells play a targeted killing role through ADCC action of the chimeric gene Fc gamma RIIIIa-BB-zeta.

Example 10 preparation of Fc γ RIIa-BB-zeta-NK (production cell preparation for allogeneic therapy).

1. Culturing NK cells from PBMC by IL-15 and IL-21 cytokine culture method, wherein the purity of the NK cells is more than 90% after 14-21 days of culture;

2. harvesting cultured NK cells, and washing three times with DPBS;

3. adding physiological saline for resuspension, and adjusting cell concentration to 5 × 10⁷Per ml, 0.1. mu.g/ml of therapeutic monoclonal antibody is added and incubated for 45 minutes at 4 ℃;

4. centrifuging at 1500rpm for 10min to collect cells after antibody incubation, adding cell freezing medium containing 10% DMSO, and adjusting cell concentration to 5 × 10⁷Placing the cells in a cell freezing bag, and transferring the cells to liquid nitrogen for storage after overnight in an ultralow temperature refrigerator;

5. when the cell needs to be transfused, liquid nitrogen or dry ice is transported to a transfusing place, is quickly thawed in a water bath kettle at 37 ℃, and then is transfused back to the body of a patient;

6. cell function verification after cryopreservation recovery: the results of the cell viability and killing ability tests are shown in FIGS. 9-10.

Fig. 9 shows that the cell survival rate after cryopreservation recovery is slightly reduced, and can still reach over 90 percent, so that the requirement of clinical treatment can be met.

FIG. 10 shows that NK cells after cryopreservation and recovery still retain the ADCC effect ability of the monoclonal antibody, the cell killing ability is slightly reduced, and the cell killing ability level can meet the requirement of clinical treatment. Can be directly returned to the patient after resuscitation, and can play the role of ADCC combined with the therapeutic monoclonal antibody.

It can be seen from the above embodiments that, on the basis of optimizing the CAR molecular structure, the CAR molecule with Fc γ rliiia as a designed action site adopts the method of directly connecting the Fc γ rliiia extracellular region with the CD8 α transmembrane region, and deletes the unique design of the CD8 α hinge region, so that the CAR molecule can be used in combination with various monoclonal antibody drugs for treating various tumors, and simultaneously, compared with the structure containing the CD8 α hinge region, the CAR molecule is more beneficial to activating effector cells and can further exert the efficient killing function of the CAR molecule on tumor cells; meanwhile, the designed immune cells of the chimeric Fc gamma RIIa gene are returned to the body of a patient after being amplified in vitro, functional ADCC effector cells are supplemented, tumor cells are specifically identified under the mediation of monoclonal antibody drugs, and the CAR molecules have the effect of killing the tumor cells.

The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

The sequence table of the chimeric gene: nucleotide and amino acid sequence listing

Sequence identifier: sequence name

SEQ ID NO 1 Fc γ RIIa-BB ζ (nucleic acid sequence)

SEQ ID NO 2 Kozak sequence (nucleic acid sequence)

3 Fc gamma RIIa Signal peptide (nucleic acid sequence)

4 Fc γ RIIa extracellular region (nucleic acid sequence)

5 CD8 alpha transmembrane sequence (nucleic acid sequence)

SEQ ID NO 64-1 BB (nucleic acid sequence)

SEQ ID NO 7 CD3 ζ (nucleic acid sequence)

8 Fc. gamma. RIIa Signal peptide (amino acid sequence) of SEQ ID NO

9 Fc γ RIIa extracellular region (amino acid sequence)

10 CD8 alpha transmembrane sequence (amino acid sequence)

SEQ ID NO 114-1 BB (amino acid sequence)

SEQ ID NO 12 CD3 ζ (amino acid sequence)

SEQUENCE LISTING

<110> Jiangsu Puruikang biomedical science and technology Co., Ltd

<120> Fc gamma RIIa-based chimeric gene and use thereof

<130>1

<160>1

<170>PatentIn version 3.3

<210>1

<211>1269

<212>DNA

<213> Artificial sequence

<400>1

gccaccatgg gtggaggggc tggggaaagg ctgtttactt cctcctgtct agtcggtttg 60

gtccctttag ggctccggat atctttggtg acttgtccac tccagtgtgg catcatgtgg 120

cagctgctcc tcccaactgc tctgctactt ctagtttcag ctggcatgcg gactgaagat 180

ctcccaaagg ctgtggtgtt cctggagcct caatggtaca gggtgctcga gaaggacagt 240

gtgactctga agtgccaggg agcctactcc cctgaggaca attccacaca gtggtttcac 300

aatgagagcc tcatctcaag ccaggcctcg agctacttca ttgacgctgc cacagtcgac 360

gacagtggag agtacaggtg ccagacaaac ctctccaccc tcagtgaccc ggtgcagcta 420

gaagtccata tcggctggct gttgctccag gcccctcggt gggtgttcaa ggaggaagac 480

cctattcacc tgaggtgtca cagctggaag aacactgctc tgcataaggt cacatattta 540

cagaatggca aaggcaggaa gtattttcat cataattctg acttctacat tccaaaagcc 600

acactcaaag acagcggctc ctacttctgc agggggcgtt ttgggagtaa aaatgtgtct 660

tcagagactg tgaacatcac catcactcaa ggtttggcag tgtcaaccat ctcatcattc 720

tttccacctg ggatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 780

tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 840

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 900

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 960

cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1020

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagcagaga 1080

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1140

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1200

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1260

cctcgctaa 1269

<210>2

<211>6

<212>DNA

<213> Artificial sequence

<400>2

gccacc 6

<210>3

<211>159

<212>DNA

<213> Artificial Synthesis

<400>3

atgggtggag gggctgggga aaggctgttt acttcctcct gtctagtcgg tttggtccct 60

ttagggctcc ggatatcttt ggtgacttgt ccactccagt gtggcatcat gtggcagctg 120

ctcctcccaa ctgctctgct acttctagtt tcagctggc 159

<210>4

<211>567

<212>DNA

<213> Artificial sequence

<400>4

atgcggactg aagatctccc aaaggctgtg gtgttcctgg agcctcaatg gtacagggtg 60

ctcgagaagg acagtgtgac tctgaagtgc cagggagcct actcccctga ggacaattcc 120

acacagtggt ttcacaatga gagcctcatc tcaagccagg cctcgagcta cttcattgac 180

gctgccacag tcgacgacag tggagagtac aggtgccaga caaacctctc caccctcagt 240

gacccggtgc agctagaagt ccatatcggc tggctgttgc tccaggcccc tcggtgggtg 300

ttcaaggagg aagaccctat tcacctgagg tgtcacagct ggaagaacac tgctctgcat 360

aaggtcacat atttacagaa tggcaaaggc aggaagtatt ttcatcataa ttctgacttc 420

tacattccaa aagccacact caaagacagc ggctcctact tctgcagggg gcgttttggg 480

agtaaaaatg tgtcttcaga gactgtgaac atcaccatca ctcaaggttt ggcagtgtca 540

accatctcat cattctttcc acctggg 567

<210>5

<211>72

<212>DNA

<213> Artificial sequence

<400>5

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210>6

<211>126

<212>DNA

<213> Artificial sequence

<400>6

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210>7

<211>339

<212>DNA

<213> Artificial sequence

<400>7

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagcagaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210>8

<211>53

<212>PRT

<213> Artificial Synthesis

<400>8

Met Gly Gly Gly Ala Gly Glu Arg Leu Phe Thr Ser Ser Cys Leu Val

1 5 10 15

Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu Val Thr Cys Pro Leu

20 25 30

Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu

35 40 45

Leu Val Ser Ala Gly

50

<210>9

<211>189

<212>PRT

<213> Artificial Synthesis

<400>9

Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln

1 5 10 15

Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly

20 25 30

Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser

35 40 45

Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val

50 55 60

Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser

65 70 75 80

Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala

85 90 95

Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His

100 105 110

Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly

115 120 125

Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys

130 135 140

Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Val Phe Gly

145 150 155 160

Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly

165 170 175

Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly

180 185

<210>10

<211>22

<212>PRT

<213> Artificial Synthesis

<400>10

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

1 5 10 15

Val Ile Thr Leu Tyr Cys

20

<210>11

<211>42

<212>PRT

<213> Artificial Synthesis

<400>11

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210>12

<211>112

<212>PRT

<213> Artificial Synthesis

<400>12

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

Claims

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;