CN117924507A - CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof - Google Patents
CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof Download PDFInfo
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Abstract
The invention discloses a CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof, wherein a connecting peptide is sequentially connected with VL αGPC3、VHαGPC3,VHαCD3 and VL αCD3,VLαGPC3 nitrogen ends and is connected with VH αGPC3 nitrogen ends, VH αGPC3 carbon end is connected with VH αCD3 carbon end, VH αCD3 nitrogen end is connected with VL αCD3 nitrogen end or VL αGPC3 carbon end is connected with VH αGPC3 carbon end, VH αGPC3 nitrogen end is connected with VH αCD3 nitrogen end, VH αCD3 carbon end is connected with VL αCD3 carbon end, and different from traditional BiTE, the light/heavy chain amino acid sequence of scFv is the same as the original sequence but opposite in translation direction through genetic engineering rearrangement of protein coding gene codon sequence, and the formed BiTE structure is more similar to a natural antibody molecular structure and is used for preparing antitumor drugs.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a CD3 epsilon and GPC3 dual-specificity T cell adapter and application thereof.
Background
Chimeric antigen receptor T cells (CHIMERIC ANTIGEN receptor-Tcell, CAR-T) are prepared by chimeric single-chain variable domain fragments (scFv) with T cell surface receptors on T cells, thereby allowing T cells to recognize tumor cells independent of major histocompatibility complex I (major histocompatibility complexI, mhc I), and then purifying, amplifying and activating in vitro, and infusing back CAR-T cells into patients, thereby allowing them to specifically exert the function of killing tumor cells. The CAR vector is mainly composed of 3 parts: an antigen binding domain, a transmembrane region, and an intracellular signaling region.
In CAR-T molecules, the intracellular signaling domain plays an important role. The effect of co-stimulation of CARs in CAR engineering is of great concern, with the goal of generating CAR constructs with optimal intracellular domains. Both of the two most common FDA-approved co-stimulatory domains CD28 and 4-1BB (CD 137) are associated with higher patient response rates. There was a difference in CD28 and 4-1BB functions. CD28 mainly promotes CAR-T proliferation, enhancing effector functions, especially cytokine production. 4-1BB promotes survival and increases retention time in CAR-T. Clinical results indicate that the CAR-T cells have great advantages in treating hematological malignancies.
The selection of tumor targets is a key factor in determining success or failure of CAR-T treatment, and the selected tumor-specific antigens need to be highly expressed in tumor tissues but not expressed or at very low expression levels in important tissues.
Glypican-3 (GPC 3) is a member of the family of heparan sulfate glycoproteins (heparan sulfate proteoglycans, HSPG), the GPC3 protein consists of 580 amino acids and has a relative molecular mass of about 70kDa. GPC3 is expressed abundantly in fetuses, whereas it is not expressed or expressed in very low amounts in most tissues of healthy adults. In contrast, immunohistochemical staining of tumor tissue in 147 HCC patients found that up to 87% (128/147) samples were GPC3 positive. Numerous documents indicate that GPC3 is an important index for HCC diagnosis and prognosis effect evaluation, and is also an ideal target for HCC treatment.
Bispecific T cell adaptors (bispecific T-CELL ENGAGER, biTE) represent a class of bispecific antibodies with remarkable anti-tumor effects, which are capable of targeted activation of self T cells to kill tumor cells. BiTE consists of two single-chain variable fragments (scFv) connected in series by a flexible linker. One scFv recognizes the T cell surface protein CD3 epsilon, while the other scFv recognizes a specific tumor cell surface antigen. This structure of BiTE and the ability to specifically bind proteins allows it to physically bridge T cells to tumor cells to form T cell-BiTE-tumor cell complexes, induce immune synapse formation, stimulate T cell activation, and produce cytokines that kill tumors. In recent years, biTE has been significantly advanced in anti-tumor studies, and clinically, an ideal therapeutic effect has been achieved.
Conventional scFv are generally designed according to the following principles:
1. Extracting a variable region fragment Fv of the IgG antibody according to the sequence, wherein the variable region fragment Fv comprises two peptide chains, namely a heavy chain variable region VH and a light chain variable region VL;
2. Linking VH and VL together through a linker to form a single peptide chain (VH-VL or VL-VH);
3. (Gly 4Ser)3 is one of the most commonly used connecting peptides at present, which is soft but the prior art has the following defects that 1) (G 4S)3 connecting peptide is too short, so that the spatial conformation of the connected scFv is far away from that of a natural antibody structure, and 2) the existing scFv targeting GPC3 has limited killing effect on tumor cells.
Disclosure of Invention
The present invention aims to overcome the disadvantages of the prior art and to provide a CD3 epsilon and GPC3 bispecific T cell adaptor and uses thereof.
The aim of the invention is achieved by the following technical scheme: a CD3 epsilon and GPC3 bispecific T cell adaptor comprising an alpha GPC3 light chain variable region VL, an alpha GPC3 heavy chain variable region VH, an alpha CD3 light chain variable region VL connected in sequence by a Linker peptide Linker, the nitrogen end of VL αGPC3 connected to the nitrogen end of VH αGPC3, the carbon end of VH αGPC3 connected to the carbon end of VH αCD3, the nitrogen end of VH αCD3 connected to the nitrogen end of VL αCD3.
Further, the nucleotide sequence of the Linker is shown as SEQ ID No.1, the nucleotide sequence of the light chain variable region VL of alpha GPC3 is shown as SEQ ID No.2, the nucleotide sequence of the light chain variable region VH of alpha GPC3 is shown as SEQ ID No.3, the nucleotide sequence of the heavy chain variable region VH of alpha CD3 is shown as SEQ ID No.4, and the nucleotide sequence of the light chain variable region VL of alpha CD3 is shown as SEQ ID No. 5.
A nucleic acid molecule comprising the CD3 epsilon and GPC3 bispecific T cell adaptors described above.
An expression vector comprising the nucleic acid molecule described above.
Further, the expression vector is a secretory expression vector, the expression plasmid is an enhanced muscle specific expression plasmid pEMS, the promoter of pEMS is EMS, and the nucleotide sequence of the pEMS is shown as SEQ ID No. 6.
Further, it also comprises a mouse Ig kappa signal peptide, and the nucleotide sequence of the mouse Ig kappa signal peptide is shown as SEQ ID No. 7.
A cell line comprising the expression vector described above.
A method of constructing a cell line comprising the steps of:
s1, synthesizing target gene fragments of CD3 epsilon and GPC3 dual-specificity T cell adaptors;
S2, cloning and connecting the target gene fragment constructed in the step S1 to pLVX-Puro vectors to form transfection plasmids;
s3, co-transferring psPAX plasmids, pMD2.G and the transfection plasmids into HEK293T cells to obtain virus liquid;
s4, infecting CHO-K1 cells by using virus liquid, and screening cell lines with stable expression by using puromycin to obtain CHO-K1 cell lines with stable expression transfection plasmid.
The application of the CD3 epsilon and GPC3 dual-specificity T cell adapter, the nucleic acid molecule, the expression vector and the cell strain in preparing antitumor drugs.
Further, the tumor is liver cancer.
The invention has the following advantages: the CD3 epsilon and GPC3 dual-specificity T cell adapter changes the connection mode of the traditional BiTE, rearranges the codon sequence of the protein coding gene by a genetic engineering technology, ensures that the light/heavy chain amino acid sequence of scFv is completely the same as the original sequence but has opposite translation directions, the formed BiTE structure is more similar to the conformation of a natural antibody molecule, the expression of therapeutic protein can be detected 7-14 days after plasmid injection is carried out on skeletal muscle, the expressed protein can activate and maintain the activity of the T cell and promote the proliferation of the T cell, and the CD3 epsilon and GPC3 dual-specificity T cell adapter can simultaneously identify and link tumor cells and T cells, guide the T cells to kill the liver cancer cells and has good killing effect on tumors; the invention utilizes skeletal muscle to secrete therapeutic protein, modifies T cells of a patient in vivo, avoids graft versus host reaction (graft versus host reaction, GVHR) and host versus graft reaction (host versus graft reaction, HVGR) caused by allogeneic CAR-T therapy, and reduces the anti-tumor treatment cost.
Drawings
FIG. 1 shows a pcDNA3.1 (+) vector map.
FIG. 2 is a schematic diagram of pEMS-mαGPC3 xαCD3 plasmids.
FIG. 3 is a block diagram of pEMS-mαGPC3 xαCD3-Ctrl and pEMS-mαGPC3 xαCD3-Exp1, wherein A is the nitrogen terminal of VL αGPC3 linked to the nitrogen terminal of VH αGPC3, the carbon terminal of VH αGPC3 linked to the carbon terminal of VH αCD3, and the nitrogen terminal of VH αCD3 linked to the nitrogen terminal of VL αCD3; the carbon terminal of VL αGPC3 is connected to the carbon terminal of VH αGPC3, the nitrogen terminal of VH αGPC3 is connected to the nitrogen terminal of VH αCD3, and the carbon terminal of VH αCD3 is connected to the carbon terminal of VL αCD3.
FIG. 4 is a pLVX-Puro vector map.
FIG. 5 is a graph showing the results of in vitro killing ability of 2 BiTEs against Hepa 1-6 cells.
FIG. 6 is a graph showing the results of 2 BiTE-induced release of IFN-gamma from PBMC.
FIG. 7 is a graph showing the results of 2 BiTE-induced PBMC release of TNF- α.
FIG. 8 is a schematic of in situ plasmid injection in mice.
FIG. 9 is a graph showing tumor growth after in situ injection of tumor-bearing mouse plasmids.
FIG. 10 is a graph showing survival after in situ injection of tumor-bearing mouse plasmids.
Detailed Description
The invention will be further described with reference to the accompanying drawings and examples, to which the scope of the invention is not limited: example 1: a CD3 epsilon and GPC3 bispecific T cell adaptor comprising an alpha GPC3 light chain variable region VL, an alpha GPC3 heavy chain variable region VH, an alpha CD3 light chain variable region VL connected in sequence by a Linker peptide Linker, the nitrogen terminus of VL αGPC3 being connected to the nitrogen terminus of VH αGPC3, the carbon terminus of VH αGPC3 being connected to the carbon terminus of VH αCD3, the nitrogen terminus of VH αCD3 being connected to the nitrogen terminus of VL αCD3, as shown as a in fig. 3; or the carbon terminal of VL αGPC3 is linked to the carbon terminal of VH αGPC3, the nitrogen terminal of VH αGPC3 is linked to the nitrogen terminal of VH αCD3, and the carbon terminal of VH αCD3 is linked to the carbon terminal of VL αCD3, as shown in B in fig. 3.
The nucleotide sequence of the connecting peptide Linker is shown as SEQ ID No. 1. The nucleotide sequence of the alpha GPC3 light chain variable region VL is shown as SEQ ID No.2, the nucleotide sequence of the alpha GPC3 light chain variable region VH is shown as SEQ ID No.3, the nucleotide sequence of the alpha CD3 heavy chain variable region VH is shown as SEQ ID No.4, and the nucleotide sequence of the alpha CD3 light chain variable region VL is shown as SEQ ID No. 5.
The method comprises the following steps:
1. linker peptide Linker: (Gly 4Ser)3 total 45bp, sequence as follows (SEQ ID No. 1):
5’-GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC-3’
2. Light chain variable region (VL αGPC3) of the targeted mouse GPC3 antibody: the total length is 336bp, the sequence is as follows (SEQ ID No. 2):
5'-GACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAGCAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAACACCCACGTGCCCCCCACCTTCGGCAGCGGCACCAAGCTGGAGATCAAG-3'
3. The heavy chain variable region (VH αGPC3) of the targeted mouse GPC3 antibody was 345bp in total length, with the following sequence (SEQ ID No. 3):
5'-CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAAGCAGACCCCCGTGCACGGCCTGAAGTGGATCGGCGCCCTGGACCCCAAGACCGGCGACACCGCCTACAGCCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGTTCTACAGCTACACCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC-3'
4. the heavy chain variable region (VH αCD3) of the targeted mouse CD3 antibody is 348bp in total, and the sequence is as follows (SEQ ID No. 4):
5'-GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCAAGAGCCTGAAGCTGAGCTGCGAGGCCAGCGGCTTCACCTTCAGCGGCTACGGCATGCACTGGGTGAGGCAGGCCCCCGGCAGGGGCCTGGAGAGCGTGGCCTACATCACCAGCAGCAGCATCAACATCAAGTACGCCGACGCCGTGAAGGGCAGGTTCACCGTGAGCAGGGACAACGCCAAGAACCTGCTGTTCCTGCAGATGAACATCCTGAAGAGCGAGGACACCGCCATGTACTACTGCGCCAGGTTCGACTGGGACAAGAACTACTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC-3'
5. The total length of the light chain variable region (VL αCD3) of the targeted mouse CD3 antibody was 321bp, and the sequence was as follows (SEQ ID No. 5):
5'-GACATCCAGATGACCCAGAGCCCCAGCAGCCTGCCCGCCAGCCTGGGCGACAGGGTGACCATCAACTGCCAGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAACAAGCTGGCCGACGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCAGGGACAGCAGCTTCACCATCAGCAGCCTGGAGAGCGAGGACATCGGCAGCTACTACTGCCAGCAGTACTACAACTACCCCTGGACCTTCGGCCCCGGCACCAAGCTGGAGATCAAG-3'
EMS is an enhanced skeletal muscle cell-specific promoter (Enhanced Muscle Specific Promoter) and is named EMS, in order to be able to help the plasmid expressed proteins secrete out of the cell, we have added a mouse Ig kappa signal peptide after the promoter and before the multiple cloning site region to direct the secretion of the expressed proteins out of the cell.
(6) EMS total length 701bp, sequence as follows (SEQ ID No. 6):
5'-GGTACCTTGATGTACTGCCAAGTTGGAAAGTCCCGTTAGTGCCCATTGACGTCAATAATATATGGCGACGGCCGGGCCCCTCCCTGGGGACAGCCCCGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGACTATATAAAAAACCTGACCCGATATGCCTGGCCAGCCAATAGCGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGACACCCAAATATGGCGACGGGTGAGGAATGGTGACCAAGTCAGCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCACCAACACCTGCTGCCTGCCCGCTCTAAAAATAACTCCCGGCTTCAGGTTTCCCTAGGGCCCCTCCCTGGGGACAGCCCCATATGGCGACGGCCCCCCATTGACGTCAATGGGACGGTAAATGGCCCGCCTGGCGCCCATTGACGTCAATAATCCAGCCAATAGCACCCGATATGCCTGGGGACTATATAAAAAACCTGGGACACCCGAGATGCCTGGTTACAAGGCCTGGGGACACGCTCTAAAAATAACTCCCCCAACACCTGCTGCCTGCCGGCTTCAGGTTTCCCTACTCGAG-3'
(7) The total length of the mouse Ig kappa signal peptide is 60bp, and the sequence is as follows (SEQ ID No. 7):
5’-GAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC-3’
The 7 parts are synthesized by Shanghai, constructed on a pcDNA3.1 (+) vector, the vector map is shown in figure 1, and the enzyme cutting sites are BamH I and EcoR I; the connection sequence is performed in the sequence shown in fig. 2. The above genes were cloned in pEMS for expression of secreted proteins.
Example 2: construction of plasmids expressing double-targeting adapter antibodies recognizing T cells and tumor cells
The BiTE comprises an antibody alpha CD3 for recognizing a mouse T cell surface antigen CD3 and an antibody alpha GPC3 for recognizing a mouse liver cancer cell surface antigen GPC3, which are connected through peptide segments. The BiTE acts to simultaneously recognize and link two cells to direct T cells to kill tumor cells. The plasmids expressing the BiTE were designated pEMS-mαGPC3 xαCD3-Ctrl, pEMS-mαGPC3 xαCD3-Exp1, pEMS-mαGPC3 xαCD3-Ctrl and pEMS-mαGPC3 xαCD3-Exp1 structures as shown in FIG. 3.
To construct CHO-K1 cell lines capable of stably expressing pEMS-mαGPC3 xαCD3-Ctrl and pEMS-mαGPC3 xαCD3-Exp 1. The present invention is characterized in that mαGPC3 xαCD3-Ctrl and mαGPC3 xαCD3-Exp1 target gene fragments are connected to pLVX-Puro vector by molecular cloning, and pLVX-Puro vector map is shown in FIG. 4.
Example 3: the lentivirus packaging steps are as follows:
1) Inoculating HEK293T cells in a 10cm dish until the cell confluency reaches 70% -90%;
2) HEK293T cell medium (10% FBS, DMEM medium containing 100IU/mL penicillin and 100. Mu.g/mL streptomycin) was replaced with 10mL fresh Quan Pei (10% FBS, 100IU/mL penicillin and 100. Mu.g/mL streptomycin DMEM medium) 1h in advance;
3) Viral packaging plasmid (psPAX and pMD2. G), transfection plasmid (pLVX-mαGPC3 αCD3-Ctrl, pLVX-mαGPC3 αCD3-Exp 1) were prepared as shown in Table 1, added to 750. Mu.L Opti-MEM, mixed, and left to stand for 5min;
Table 1: preparation of lentivirus packaging plasmid
4) Adding 24 μL of Lipo8000 into 750 μL of Opti-MEM, mixing, and standing for 5min;
5) Mixing the solutions obtained in the steps 3 and 4, and incubating for 5min at room temperature;
6) Uniformly dispersing the mixed solution obtained in the step 5 into HEK293T cells, uniformly shaking in a cross manner, and putting into an incubator;
7) After 12h, 10mL of complete medium (DMEM medium containing 10% FBS, 100IU/mL penicillin and 100. Mu.g/mL streptomycin) was changed, and calculation was started from this point. After 48 hours, collecting the cell culture solution (namely virus solution) into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 minutes;
8) The supernatant was aspirated by syringe, filtered through 0.45 μm microporous filter membrane, dispensed into 1.5mL EP tubes, 500. Mu.L per tube, sealed with sealing membrane and stored at-80 ℃.
Example 4: CHO-K1 cell lentiviral infection:
1) And (3) paving: spreading a 6-hole plate to be infected with cells CHO-K1 until the cells grow to 60-80%;
2) Changing 600 μl of complete culture medium, sequentially adding 500 μl of virus solution, and polybrene (final concentration 10 μg/mL); incubating the incubator overnight;
3) The following day, the complete medium was changed and after 48h, screened with puromycin (final concentration 2. Mu.g/mL);
example 5: stable expression cell strain selection:
1) After 72h, adding puromycin with a final concentration of 2 mug/mL for culturing for 24h, changing to normal culture medium if the cell confluency is lower than 30%, culturing until the cell confluency is higher than 30%, changing to fresh culture medium with a concentration of 8 mug/mL puromycin (10% FBS, 100IU/mL penicillin, 100 mug/mL streptomycin and 8 mug/mL DMEM culture medium) for culturing and screening for 2 weeks; if the cell confluency is not less than 30%, 2 mug/mL puromycin fresh medium is used for continuous culture screening for 2 weeks;
2) The obtained CHO-K1 cell lines stably expressing pLVX-mαGPC3 xαCD3-Ctrl and pLVX-mαGPC3 xαCD3-Exp1 were frozen at-80℃for use.
Example 6: the effect of expressed antibodies on T cell killing capacity was studied:
mice Hepa 1-6 were labeled with 1 μ L CELL TRACKER Violet and tumor cells after labeling were incubated overnight at 37 ℃. After obtaining peripheral blood mononuclear cells (PERIPHERAL BLOOD MONOCYTE CELL, PBMC) from mice, enriched lymphocytes were incubated overnight at 37℃at a concentration of 1X 10 6/mL, and adherent cells were removed the next day, at which time the cells were ready for use in subsequent co-culture experiments. Target cells were co-incubated with adherent cell depleted PBMCs [ effector cells: target (E: T) cell ratio, 1:1,4:1, 10:1]. After the co-culture is completed, the upper layer of suspended lymphocytes are discarded, PBS is used for washing, and the attached target cells are digested by pancreatin and then subjected to flow cytometry analysis, as shown in FIG. 5, it can be seen from FIG. 5 that mαGPC3 xαCD3-Exp1 shows a tumor cell killing effect similar to mαGPC3 xαCD3-Ctrl.
The IFN-gamma content released by lymphocytes in PBMC is detected by ELISA kit, the operation steps are described by referring to the Eboltag kit (Cat: RK 00019), the TNF-alpha content released by lymphocytes in PBMC is detected by ELISA kit, the operation steps are described by referring to the Eboltag kit (Cat: RK 00027), and after mαGPC3 xαCD3-Exp1 treatment, more IFN-gamma is released by PBMC than after mαGPC3 xαCD3-Ctrl treatment as shown in FIG. 6; after mαGPC3 xαCD3-Exp1 treatment, the PBMC released TNF- α content was comparable to that of the mαGPC3 xαCD3-Ctrl treated group as shown in FIG. 7. From the cellular level, it was verified that mαGPC3 xαCD3-Exp1 exhibited cell killing ability comparable to that of conventional mαGPC3 xαCD3-Ctrl.
Example 7: treatment effect detection
1. Mice weight detection:
In situ plasmid injection is schematically shown in FIG. 8 (cell inoculation 6X 10 5/mouse, plasmid injection (40. Mu.g/mouse), time to kill mice, etc.); day 0 each mouse was inoculated with 6 x 10 5 Hepa 1-6 liver cancer cells, day 2 mice were injected with 40 μg plasmid, and 1h post-plasmid injection was shocked with a Datura-brand electric needle for 3min to assist plasmid nucleation (continuous wave, 5Hz, intensity 3).
2. Tumor volume detection in mice:
Tumor volume was measured every two days during animal experiments, and tumor growth curves of tumor-bearing mice were plotted, and it can be seen from fig. 9 that tumor growth of tumor-bearing mice was inhibited after mαgpc3×αcd3-Exp1 treatment, and the therapeutic effect was not significantly different from mαgpc3×αcd3-Ctrl.
3. Median survival of mice:
Counting the death time of the mice, and judging whether each treatment scheme prolongs the service life of the mice; tumor-bearing mice, which had tumor volumes exceeding 1500mm3 or mice died, were considered the experimental endpoint and tumor-bearing mice were plotted for survival, as shown in fig. 10, showing comparable survival rates after treatment with mαgpc3×αcd3-Exp1 plasmid as after treatment with mαgpc3×αcd3-Ctrl plasmid.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.
Claims (10)
1. A CD3 epsilon and GPC3 bispecific T cell adaptor comprising an alpha GPC3 light chain variable region VL, an alpha GPC3 heavy chain variable region VH, an alpha CD3 light chain variable region VL connected in sequence by a Linker peptide Linker, the nitrogen terminal of VL αGPC3 being connected to the nitrogen terminal of VH αGPC3, the carbon terminal of VH αGPC3 being connected to the carbon terminal of VH αCD3, the nitrogen terminal of VH αCD3 being connected to the nitrogen terminal of VL αCD3;
Or the carbon terminal of VL αGPC3 is linked to the carbon terminal of VH αGPC3, the nitrogen terminal of VH αGPC3 is linked to the nitrogen terminal of VH αCD3, and the carbon terminal of VH αCD3 is linked to the carbon terminal of VL αCD3.
2. The CD3 epsilon and GPC3 bispecific T cell adapter of claim 1, wherein the Linker peptide Linker has a nucleotide sequence shown in SEQ ID No. 1, the light chain variable region VL of αgpc3 has a nucleotide sequence shown in SEQ ID No.2, the light chain variable region VH of αgpc3 has a nucleotide sequence shown in SEQ ID No. 3, the heavy chain variable region VH of αcd3 has a nucleotide sequence shown in SEQ ID No. 4, and the light chain variable region VL of αcd3 has a nucleotide sequence shown in SEQ ID No. 5.
3. A nucleic acid molecule comprising the CD3 epsilon and GPC3 bispecific T cell engager of claim 1 or 2.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. The expression vector of claim 4, wherein the expression vector is a secretory expression vector, the expression plasmid is an enhanced muscle-specific expression plasmid pEMS, the promoter of pEMS is EMS, and the nucleotide sequence of pEMS is shown as SEQ ID No. 6.
6. The expression vector of claim 5, further comprising a mouse Ig kappa signal peptide having the nucleotide sequence set forth in SEQ ID No. 7.
7. A cell line comprising the expression vector of claim 4.
8. The method for constructing a cell line according to claim 7, comprising the steps of:
s1, synthesizing the target gene fragment of claim 1;
s2, cloning and connecting the target gene fragment constructed in the step S1 to pLVX-Puro vectors to form transfection plasmids;
s3, co-transferring psPAX plasmids, pMD2.G and the transfection plasmids into HEK293T cells to obtain virus liquid;
S4, infecting CHO-K1 cells by using virus liquid, and screening cell strains with stable expression by using puromycin to obtain CHO-K1 cell strains with stable expression transfection plasmid.
9. Use of CD3 epsilon and GPC3 bispecific T cell adaptors according to claim 1 or 2, a nucleic acid molecule according to claim 3, an expression vector according to claim 4 or 5 or 6, a cell strain according to claim 7 for the preparation of an antitumor drug.
10. The use according to claim 9, wherein the tumor is liver cancer.
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