CN117264063A

CN117264063A - anti-GPC 3 antibody and application thereof

Info

Publication number: CN117264063A
Application number: CN202310281955.8A
Authority: CN
Inventors: 翟晓晨; 李茵; 彭延; 王雅萍
Original assignee: Cyagen Biosciences Inc Suzhou
Current assignee: Cyagen Biosciences Inc Suzhou
Priority date: 2023-03-22
Filing date: 2023-03-22
Publication date: 2023-12-22
Anticipated expiration: 2043-03-22
Also published as: CN117264063B

Abstract

The invention belongs to the field of biological medicine, and particularly discloses an anti-GPC 3 antibody and application thereof. According to the invention, an antibody sequence of highly targeted GPC3 is selected, the GPC 3-targeted CAR-NK cell is designed and constructed by using the antibody sequence, experiments prove that the GPC3-CAR-NK cell has good anti-tumor activity on a liver cancer tumor cell line, has potential for preparing biological medicines for treating liver cancer, and provides a potential treatment method for liver cancer patients.

Description

anti-GPC 3 antibody and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly discloses an anti-GPC 3 antibody and application thereof, and relates to preparation and application of NK cells modified by a GPC3 chimeric antigen receptor.

Background

Chimeric antigen receptor-modified T cell (CAR-T) therapy is a novel method of immune cell therapy, and is also known as one of the representative techniques of "cellular immunotherapy". The therapy is to reform T cells to make them have specific recognition capability to tumor cells and to proliferate and attack cancer cells in large quantity in vivo so as to achieve the purpose of treating tumor. Wherein the Chimeric Antigen Receptor (CAR) is an artificially constructed antigen receptor comprising the structure of 3 parts: an extracellular domain, a transmembrane region, and an intracellular domain. The extracellular domain is generally composed of scFv and hinge regions consisting of antibody-derived light and heavy chain variable regions, the function of scFv being to incubate CAR engineered cell targeting; the transmembrane region is typically the transmembrane region of a CD8 a or CD28 molecule, which serves primarily to anchor the CAR molecule to the cell membrane; the intracellular region is composed of a costimulatory domain (e.g., 4-1BB or CD28, etc.) and a CD3ζ signaling region.

CAR-T cell therapy has achieved significant results in the treatment of hematological neoplasms at present, and a number of CAR-T cell therapeutic products are currently marketed. However, CAR-T cell therapy still faces many difficulties, such as serious toxic side effects (cytokine storm, neurotoxicity, etc.), high individuality, difficult industrialization, poor therapeutic effect of solid tumors, etc. Therefore, in recent years, engineering other immune cell therapies has also become a very popular direction of research, including the study of CAR-NK cells.

Natural killer cells (NK cells) are an innate immune cell that plays an important role in the body's resistance to pathogen infection and tumors. NK cells function most similarly to CD 8T cells, but unlike T cells, NK cells are a class of lymphocytes that can kill tumor cells and virus-infected cells non-specifically without prior sensitization. The lysis of NK cells to tumor cells is not limited by MHC molecules, so NK cells can also be used for allogeneic therapy, and the defect that the industrialization of CAR-T cell therapy is difficult to realize can be overcome. Meanwhile, NK cell therapy can overcome the defect of strong toxic and side effects of CAR-T cell therapy. For the above reasons, NK cells have also become currently the effector cells of the intense research for immunotherapy.

Liver cancer (Liver cancer) refers to a malignant tumor that occurs in or begins with the Liver. Primary liver cancer is the second highest frequency of cancer (6%) and the sixth highest mortality (9%). At present, the means for treating liver cancer mainly comprise operation, radiotherapy and chemotherapy, but the treatment effect is not ideal, and the five-year survival rate of liver cancer patients is still very low. Therefore, development of new therapeutic means for liver cancer is urgent.

Disclosure of Invention

In order to solve the problems, the invention discloses an anti-GPC 3 antibody and application thereof. GPC3 is the target of liver cancer treatment, and is an abbreviation for glycoprotein conjugate 3 (Glypican-3). GPC3 is a small protein that is interposed between the extracellular matrix and the envelope and plays an important role in cell growth, differentiation, tumor metastasis, cell adhesion, and signaling. GPC3 is mainly present in liver cancer and hepatocellular carcinoma cells, but is also expressed in some malignant tumors such as pancreatic cancer and ovarian cancer. As a marker for liver cancer, GPC3 has become one of the important targets for liver cancer treatment.

The technical scheme of the invention is as follows:

an anti-GPC 3 antibody having the amino acid sequence of the light chain variable region VL of SEQ ID NO:3 is shown in the figure; the amino acid sequence of the heavy chain variable region VH of the antibody is shown in SEQ ID NO: shown at 7.

Further, a chimeric antigen receptor comprising the amino acid sequence of the light chain variable region or heavy chain variable region of the above antibody.

Further, a nucleic acid molecule encoding the amino acid sequence of the light chain variable region of the above antibody or the amino acid sequence of the heavy chain variable region of the antibody; or an amino acid sequence encoding the above antibody; or an amino acid sequence encoding the chimeric antigen receptor described above.

Further, a vector, the nucleic acid molecule.

Further, an antibody conjugate comprising a) an antibody moiety comprising the above antibody or a variable region of the antibody or said chimeric antigen receptor; b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.

Further, an engineered cell can express the antibody, the variable region of the antibody, or the chimeric antigen receptor.

Further, the preparation method of the engineering cell comprises the following steps: the above-described nucleic acid molecules or vectors are transduced into host cells to thereby obtain the engineered cells.

Further, in the preparation method of the engineering cell, the host cell is an NK cell.

Furthermore, the antibody variable region, the nucleic acid, the vector or the engineering cell can be applied to the development of medicaments for treating liver cancer.

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

according to the invention, an antibody sequence of highly targeted GPC3 is selected, the GPC 3-targeted CAR-NK cell is designed and constructed by using the antibody sequence, experiments prove that the GPC3-CAR-NK cell has good anti-tumor activity on a liver cancer tumor cell line, has potential for preparing an immune medicament for treating liver cancer, and provides a potential treatment method for liver cancer patients.

Drawings

FIG. 1.GPC3 antigen lentiviral vector map;

FIG. 2. Detection of the positive rate of GPC3-CHO-K1 stable cell line;

FIG. 3 construction of LV-GPC3CAR-MCS-kana vector map according to the present invention;

FIG. 4 statistics of fold expansion of NK cells;

FIG. 5 purity detection of NK cells;

FIG. 6. Detection of the positive rate of GPC3 CAR-NK;

FIG. 7 shows measurement of expression level of GPC3 antigen on HuH7 cell surface;

FIG. 8 shows the lytic effect of GPC3 CAR-NK cells on human liver cancer Huh7 cells;

FIG. 9 IFN-. Gamma.secretion assay of GPC3 CAR-NK cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise specified, the various reagents involved in the examples are all commercially available: the specific procedures referred to in the examples are described in the third edition of the guidelines for molecular cloning experiments, unless otherwise specified.

The nucleic acid or amino acid sequences mentioned in the present invention are as follows

SEQ ID NO 1：

MLLLVTSLLLCELPHPAFLLIP

SEQ ID NO 2：

ATGCTGCTCCTGGTGACAAGCCTGCTCCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATCCCC

SEQ ID NO 3：

DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLRINSLQPEDFGSYSCQHFWSTPPYTFGGGTKLEIK

SEQ ID NO 4：

GACATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCACCTGCAGAGCTAGCGGCAACATCCACAACTACCTGGCCTGGTATCAGCAGAAGCAAGGCAAGAGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGACGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACACAGTACAGCCTGAGAATCAACAGCCTGCAGCCCGAGGACTTCGGCAGCTACAGCTGTCAGCACTTCTGGAGCACCCCCCCCTACACCTTCGGCGGGGGGACCAAGCTGGAAATCAAG

SEQ ID NO 5：

GGGGSGGGGSGGGGS

SEQ ID NO 6：

GGCGGGGGCGGGAGCGGGGGGGGGGGGAGCGGCGGGGGCGGCAGC

SEQ ID NO 7：

QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSSSYTKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARSATVGAMDYWGQGTSVTVSS

SEQ ID NO 8：

CAAGTGCAGCTCCAACAGTCCGGCGCCGAGCTGGCCAAGCCCGGCGCTAGCGTGAAGCTGAGCTGCAAGGCTAGCGGCTACACCTTCACAAGCTACTGGATGCACTGGGTGAAGCAGAGACCCGGCCAAGGCCTGGAGTGGATCGGCTACATCAACCCTAGCAGCAGCTACACCAAGTACAATCAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCTACGAGGACAGCGCCGTGTACTACTGCGCTAGAAGCGCCACCGTGGGCGCCATGGACTACTGGGGCCAAGGCACAAGCGTGACCGTGAGCAGC

SEQ ID NO 9：

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

SEQ ID NO 10：

GAGAGCAAATACGGCCCCCCTTGCCCCCCCTGTCCCGCTCCCGAATTCCTGGGCGGCCCTAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTCGTGGACGTGAGCCAAGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCTAGAGAGCCCCAAGTGTACACCCTCCCCCCTAGCCAAGAGGAGATGACCAAGAACCAAGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGGCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCTGGGCAAGATG

SEQ ID NO 11：

FWVLVVVGGVLACYSLLVTVAFIIFWV

SEQ ID NO 12：

TTCTGGGTGCTGGTGGTCGTGGGCGGCGTGCTGGCCTGCTACAGCCTCCTCGTGACCGTGGCTTTTATCATCTTCTGGGTG

SEQ ID NO 13：

RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

SEQ ID NO 14：

AGAAGCAAGAGAAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCTAGAAGACCCGGCCCCACAAGAAAGCACTATCAGCCCTACGCCCCCCCTAGAGACTTCGCCGCCTACAGAAGC

SEQ ID NO 15：

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO 16：

AGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTATCAGCAAGGGCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCTAGAAGAAAGAACCCCCAAGAGGGCCTGTACAATGAGCTGCAAAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGACGGAGAGGCAAGGGCCACGACGGCCTGTACCAAGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAAGCCCTGCCCCCTAGATGA。

SEQ ID NO 17：

ATGGCCGGGACCGTGCGCACCGCGTGCTTGGTGGTGGCGATGCTGCTCAGCTTGGACTTCCCGGGACAGGCGCAGCCCCCGCCGCCGCCGCCGGACGCCACCTGTCACCAAGTCCGCTCCTTCTTCCAGAGACTGCAGCCCGGACTCAAGTGGGTGCCAGAAACTCCCGTGCCAGGATCAGATTTGCAAGTATGTCTCCCTAAGGGCCCAACATGCTGCTCAAGAAAGATGGAAGAAAAATACCAACTAACAGCACGATTGAACATGGAACAGCTGCTTCAGTCTGCAAGTATGGAGCTCAAGTTCTTAATTATTCAGAATGCTGCGGTTTTCCAAGAGGCCTTTGAAATTGTTGTTCGCCATGCCAAGAACTACACCAATGCCATGTTCAAGAACAACTACCCAAGCCTGACTCCACAAGCTTTTGAGTTTGTGGGTGAATTTTTCACAGATGTGTCTCTCTACATCTTGGGTTCTGACATCAATGTAGATGACATGGTCAATGAATTGTTTGACAGCCTGTTTCCAGTCATCTATACCCAGCTAATGAACCCAGGCCTGCCTGATTCAGCCTTGGACATCAATGAGTGCCTCCGAGGAGCAAGACGTGACCTGAAAGTATTTGGGAATTTCCCCAAGCTTATTATGACCCAGGTTTCCAAGTCACTGCAAGTCACTAGGATCTTCCTTCAGGCTCTGAATCTTGGAATTGAAGTGATCAACACAACTGATCACCTGAAGTTCAGTAAGGACTGTGGCCGAATGCTCACCAGAATGTGGTACTGCTCTTACTGCCAGGGACTGATGATGGTTAAACCCTGTGGCGGTTACTGCAATGTGGTCATGCAAGGCTGTATGGCAGGTGTGGTGGAGATTGACAAGTACTGGAGAGAATACATTCTGTCCCTTGAAGAACTTGTGAATGGCATGTACAGAATCTATGACATGGAGAACGTACTGCTTGGTCTCTTTTCAACAATCCATGATTCTATCCAGTATGTCCAGAAGAATGCAGGAAAGCTGACCACCACTGAAACTGAGAAGAAAATATGGCACTTCAAATATCCTATCTTCTTCCTGTGTATAGGGCTAGACTTACAGATTGGCAAGTTATGTGCCCATTCTCAACAACGCCAATATAGATCTGCTTATTATCCTGAAGATCTCTTTATTGACAAGAAAGTATTAAAAGTTGCTCATGTAGAACATGAAGAAACCTTATCCAGCCGAAGAAGGGAACTAATTCAGAAGTTGAAGTCTTTCATCAGCTTCTATAGTGCTTTGCCTGGCTACATCTGCAGCCATAGCCCTGTGGCGGAAAACGACACCCTTTGCTGGAATGGACAAGAACTCGTGGAGAGATACAGCCAAAAGGCAGCAAGGAATGGAATGAAAAACCAGTTCAATCTCCATGAGCTGAAAATGAAGGGCCCTGAGCCAGTGGTCAGTCAAATTATTGACAAACTGAAGCACATTAACCAGCTCCTGAGAACCATGTCTATGCCCAAAGGTAGAGTTCTGGATAAAAACCTGGATGAGGAAGGGTTTGAAAGTGGAGACTGCGGTGATGATGAAGATGAGTGCATTGGAGGCTCTGGTGATGGAATGATAAAAGTGAAGAATCAGCTCCGCTTCCTTGCAGAACTGGCCTATGATCTGGATGTGGATGATGCGCCTGGAAACAGTCAGCAGGCAACTCCGAAGGACAACGAGATAAGCACCTTTCACAACCTCGGGAACGTTCATTCCCCGCTGAAGCTTCTCACCAGCATGGCCATCTCGGTGGTGTGCTTCTTCTTCCTGGTGCACTGA

Example 1

Screening for GPC3 antibodies.

1 GPC3-CHO-K1 cell construction

The human GPC3 CDS region sequence (SEQ ID NO: 17) was genetically synthesized and subcloned into a lentiviral vector, the structure of which is shown in FIG. 1. After the correct sequence was verified by sequencing, the sequence was used for lentivirus preparation, and the lentivirus preparation method was referred to in example 3, item 2, "GPC3-CAR lentivirus preparation". The titer of the lentivirus is detected after the preparation is finished, and the lentivirus is used for constructing a stable cell line in GPC3-CHO-K1 after the lentivirus is qualified.

CHO-K1 cells with growth fusion rates of 80% -90% were collected and re-seeded in 24-well plates at 1×10 ⁵ And/or holes. The following day after inoculation (24 h), 30 μl of GPC3 antigen lentivirus was added to the cells. After the cells had been expanded, they were re-inoculated into 6 cm dishes and drug screened by adding puromycin. After two weeks of maintenance of the drug screen, GPC3 antigen lentiviral transfected CHO-K1 cells were collected, labeled with murine anti-human GPC3 antibody, and the GPC3 antigen positive rate was detected by an up-flow cytometer, and the results showed that: the positive rate of GPC3 antigen was nearly 100%, which indicates that we successfully constructed GPC3-CHO-K1 stable cell lines, and the results are shown in FIG. 2.

2 immunization of animals

2X 10 was intraperitoneally injected into 5 female C57 BL/6N mice each of 6-8 weeks ⁷ GPC3-CHO-K1 cells

Immunization was performed with each immunization being 2 weeks apart from the first three. On day5 after the end of the 3 rd immunization, 200. Mu.l of mouse peripheral blood was collected through the inner canthus, and the serum GPC3 antibody titer of the mouse was measured by the Elisa method. According to the detection result, the mice with the best immunization titers are selected, and the mice are subjected to booster immunization on the 10 th day after the third immunization: infusion through the tail vein 2X 10 ⁷ GPC3-CHO-K1 cells.

3 hybridoma cell fusion

Mice were euthanized 3 days after boost, spleens were dissected under sterile conditions, and spleen cells were prepared as single cell suspensions, mixed with SP/20 cells at a ratio of 1:5, and fused using PEG 400. Following fusion, cells were resuspended using DMEM medium containing 20% fetal bovine serum, 1% HAT medium, and 10% Clone easy, and subsequently plated into 96-well plates. The culture medium was changed to HT medium after 7 days of fusion and continued. On the tenth day of fusion, hybridoma cell culture supernatants were taken and assayed for antibody secretion using the Elisa method. Cells were selected for detection of wells with higher OD values and two rounds of cloning screening were performed using limiting dilution. Subcloned hybridoma cells with higher Elisa detection OD value are selected for sequencing and modulating antibody gene sequences.

Example 2

NK cell preparation process development.

NK cells in human PBMC were enriched by sorting using NK cell sorting kit (Meitian and Tsaoko, cat# 130-092-657). The principle is as follows: the cells were bound to magnetic beads by biotin-labeled antibodies (without NK cell surface molecule antibodies) by incubating with PBMC followed by the addition of magnetic beads. Then, the magnetic bead-cell mixed solution is added to a separation column placed in a magnetic field, cells bound with the magnetic beads under the action of the magnetic field are bound with the separation column, NK cells which are not bound with the magnetic beads flow out along with a buffer solution, and the cells in the collected effluent liquid are NK cells.

The NK cell preparation process flow is as follows: PBMC were incubated with Biotin-Antibody Cocktail in the kit at 4deg.C for 5min, followed by additional MicroBead Cocktail at 4deg.C for 10 min. After the co-incubation is finished, the mixture passes through an MS column in a magnetic field, and effluent liquid is collected, wherein cells in the effluent liquid are NK cells. The partially enriched NK cells were then taken, labeled with anti-human CD3 and anti-human CD56 antibodies, and analyzed for NK cell purity by flow cytometry. After the purity was acceptable, the remaining NK cells were inoculated into 24 well plates, and NK cell complete medium was supplemented to 2 ml. Subsequently, NK cell growth was periodically monitored and NK cell expansion fold was counted, and as shown in FIG. 4, this method can achieve efficient expansion of NK cells by about 2000 fold. Meanwhile, on the 14 th day of NK cell culture, cells were collected, and NK cell purity was again examined using flow cytometry, and the results showed that: the method can prepare NK cells with high purity, and the purity of the NK cells can reach more than 95% (CD 3-CD56+ cells), as shown in figure 5.

The reagents used in the process of the embodiment 2 are all reagents without animal-derived components, so that the treatment risk caused by animal-derived substances can be avoided, and the cost of quality inspection and release of products can be reduced. This preparation method can meet the needs of subsequent NK therapies for clinical use.

Example 3

GPC3-CAR-NK cell preparation

Construction of GPC3CAR lentiviral plasmid

The 10D 3CAR molecule targeting GPC3 is constructed by using the GPC3 antibody heavy chain variable region sequence and light chain variable region sequence of the 10D3 clone number obtained by screening in the invention, and the structure comprises: GM-CSF R alpha signal peptide amino acid sequence (SEQ ID NO: 1), DNA sequence (SEQ ID NO: 2); GPC3 antibody light chain variable region VL amino acid sequence (SEQ ID NO: 3), DNA sequence (SEQ ID NO: 4); linker amino acid sequence (SEQ ID NO: 5), DNA sequence (SEQ ID NO: 6); GPC3 antibody heavy chain variable region VH amino acid sequence (SEQ ID NO: 7), DNA sequence (SEQ ID NO: 8); igG4 Fc amino acid sequence (SEQ ID NO: 9), DNA sequence (SEQ ID NO: 10); CD28 transmembrane region amino acid sequence (SEQ ID NO: 11), DNA sequence (SEQ ID NO: 12); CD28 costimulatory domain amino acid sequence (SEQ ID NO: 13), DNA sequence (SEQ ID NO: 14); CD3 zeta intracellular signaling domain amino acid sequence (SEQ ID NO: 15), DNA sequence (SEQ ID NO: 16). After synthesis, subcloning the DNA sequence of the CAR molecule into LV-EF1 alpha-MCS-kana vector to construct a 10D 3CAR lentiviral plasmid; the constructed vector was designated as LV-GPC3CAR-MCS-kana, and its structure map is shown in FIG. 3. The sequence was verified to be correct for lentiviral packaging by sequencing.

In the above structure, scFv composed of VL, linker and VH can endow NKT cells with targeting, intracellular co-stimulatory domain and CD3 zeta signaling domain can provide activation signals required for activating NKT cells, thus the constructed CAR-NKT cells can have remarkably enhanced specific antitumor activity.

GPC3-CAR lentivirus preparation

Lentiviral packaging was performed by 293T cells using a third generation four plasmid lentiviral packaging system. Take 1T 175 flask as an example: (1) day 1, inoculation of about 4.5X10 ⁶ 293T cells were placed in flasks, medium 25 ml; (2) day 3, transfection; firstly, preparing 2 centrifuge tubes of 15 ml, namely a Mix A tube and a Mix B tube, adding 9.33 mug of pVSV-G, 17.5 mug of pGag/Pol, 13.42 mug of pRev, 29.63 mug of GPC3CAR lentiviral expression plasmid and 1750 mu l of DMEM basal medium into the Mix A tube, adding 140 mu l of PEI (1 mu G/mu l, polysciences, product number: 24765-100) and 1750 mu l of DMEM basal medium into the Mix B tube, mixing the Mix A tube and the Mix B tube to a block of room temperature, incubating for 10-15min, and finally adding the incubated DNA-PEI compound dropwise into a culture flask, and gently mixing; (3) day5, collecting the transfected 48 h viral supernatant into a 50 ml centrifuge tube, and supplementing 25 ml fresh medium; (4) day 6, collecting the transfected virus supernatant of 72 h into a 50 ml centrifuge tube, centrifuging the twice collected virus supernatant at 4 ℃ for 10 min at 3000 g to remove cell debris and impurities, filtering and sterilizing by using a 0.22 um small filter, and finally adding a proper amount of PEG6000 (Shanghai, cat# A610432-0500) (0.245 ml PEG6000 concentrate is added to each ml virus filtrate) for overnight concentration; (5) day 7, concentrating overnight virus at 4deg.C, centrifuging at 2000 g for 15min, discarding supernatant, adding appropriate amount of 1×HBSS (OriCell, cat# HBS-10001), and re-suspending, and purifying with sucrose (20% sucrose)VETEC, cat No.: V900116-500G), and finally re-suspending the purified lentiviral particles by using a proper amount of 1 XHBSS, sub-packaging the virus suspension into 0.5 ml freezing tubes, and storing at-80 ℃.

GPC3-CAR-NK cell preparation

Collecting NK cells amplified for 3 days, counting cells, and mixing NK cells at 1×10 ⁶ GPC3 lentivirus was calculated at moi=20 and lentivirus was added to NK cell wells to construct GPC3 CAR-NK cells. After the GPC3 CAR-NK cells were expanded, the GPC3 CAR-NK cells were collected and labeled with anti-human Fc antibodies, and the GPC3-CAR-NK positive rate was detected by flow cytometry, which showed that: the positive rate of GPC3 CAR-NK cells was 58%, as shown in FIG. 6.

Example 4

GPC3-CAR-NK cell antitumor Activity evaluation

Huh7 cells expressing EGFP were used as target cells. As shown in FIG. 7, the expression level of GPC3 antigen on the Huh7 cell surface was approximately 100% as high as that of GPC3 antigen on the Huh7 cell surface. Expression of EGFP can be lost with apoptosis of target cells, so the fraction of EGFP positive cells after killing can characterize the killing capacity of GPC3 CAR-NK cells.

To verify the lytic ability of GPC3 CAR-NK cells to tumor cells, the following experiments were designed: EGFP-Huh7 cells were first grown at 2X 10 ⁵ The cells/well were inoculated into a 48-well plate, and untransfected NK cells and GPC3 CAR-NK cells were then added to the 48-well plate at a ratio of 1:2, 1:1, 5:1, respectively, for co-culture for 24 h. After the culture is finished, the residual situation of the tumor cells is detected by the up-flow cytometer, and the result is shown in fig. 8, which shows that the GPC3 CAR-NK cells can kill and lyse the target cells Huh7 more effectively than the NK cells under different effect target ratios.

At 16 th h of co-incubation, 100 μl of co-culture supernatant from the killing experiment was collected. The results of the detection of the IFN-. Gamma.content of cells in the co-culture supernatants of each group were shown in FIG. 9, referring to IFN-. Gamma.Elisa kit (Union) detection instructions, and showed that GPC3 CAR-NK cells had significantly enhanced cytokine secretion compared to NK cells, which was consistent with the results of tumor killing experiments.

The results show that compared with NK cell GPC3-CAR-NK cells, the Huh7 cells have obviously enhanced specific killing function, and the secretion level of the cytokine IFN-gamma is far higher than that of the NK cells. Therefore, the CAR-NK cells have great application prospect in the aspect of tumor treatment.

The foregoing is a description of a limited number of preferred embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims

1. An anti-GPC 3 antibody, wherein the amino acid sequence of the light chain variable region VL of the antibody is as set forth in SEQ ID NO:3 is shown in the figure; the amino acid sequence of the heavy chain variable region VH of the antibody is shown in SEQ ID NO: shown at 7.

2. A chimeric antigen receptor comprising the amino acid sequence of the light chain variable region or the heavy chain variable region of the antibody of claim 1.

3. A nucleic acid molecule encoding the amino acid sequence of the light chain variable region of the antibody of claim 1 or the amino acid sequence of the heavy chain variable region of the antibody, or encoding the amino acid sequence of the chimeric antigen receptor of claim 3.

4. A vector comprising the nucleic acid molecule of claim 3.

5. An antibody conjugate comprising a) an antibody moiety comprising the chimeric antigen receptor of claim 1 or claim 3; b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.

6. An engineered cell expressing the antibody of claim 1; or the chimeric antigen receptor of claim 2.

7. The method of preparing an engineered cell of claim 6, comprising the steps of: transduction of the nucleic acid molecule of claim 3 or the vector of claim 4 into a host cell, thereby obtaining said engineered cell.

8. The method of claim 7, wherein the host cell is an NK cell.

9. Use of an antibody according to claim 1, or a chimeric antigen receptor according to claim 2, or an engineered cell according to claim 6, in the development of a medicament for the treatment of liver cancer.