CN111732665B - Chimeric antigen receptor of cells for targeted expression of carcinoembryonic antigen - Google Patents
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- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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Abstract
The invention relates to the technical field of biology, in particular to a chimeric antigen receptor of a cell for targeted expression of carcinoembryonic antigen. The invention also relates to nucleic acid for coding the chimeric antigen receptor, a recombinant expression vector for expressing the chimeric antigen receptor and an immune cell. The invention also relates to single chain antibodies targeting carcinoembryonic antigen. By introducing the chimeric antigen receptor into a lentivirus expression vector and then infecting immune cells, the chimeric antigen receptor targeting CEA can be expressed on the surface of the immune cells, and can effectively and specifically target malignant cells expressing CEA surface antigen, thereby providing a more efficient method with fewer side effects and adverse reactions for treating some tumors expressing CEA surface antigen.
Description
Technical Field
The invention relates to the field of cellular immunotherapy, in particular to a chimeric antigen receptor of a cell for targeted expression of carcinoembryonic antigen.
Background
Cellular immunotherapy is an emerging method for tumor therapy, which constructs an expression vector for a Chimeric Antigen Receptor (CAR) by molecular biology techniques, introduces the expression vector into immune cells isolated from a human body, expresses the CAR on the cell surface, and then performs amplification culture to return the CAR to the human body. The CAR-expressing immune cells are capable of specifically recognizing and binding to target cells, killing them by releasing specific immune factors. The first generation CAR-T, although able to mediate killing of tumor cells, does not transduce proliferation signals and induce cytokine production, and has a short duration of action in vivo, resulting in only transient T cell proliferation, and therefore, has little anti-tumor effect; the second generation CAR-T increases the intracellular structural domain of a costimulatory molecule (such as 4-1BB or CD28) to prolong the survival time in vivo and promote the rapid amplification capacity, and the CAR-T constructed by scFv/4-1BB (or CD28)/CD3-z can crack target cells, transmit activation signals and generate a large amount of cytokines such as IFN-gamma and IL-2; the second generation CAR-T has better abilities to enhance T cell activation, expansion, anti-tumor, and promote transgene expression than the first generation.
CAR-T has major advantages in killing tumors: first, since the antigen recognition binding domain thereof can recognize a tumor antigen, the specificity against a tumor is high; secondly, because the intracellular signaling domain can rapidly and simultaneously turn on downstream signals of killing and amplification of CAR-T, the tumor killing efficiency is high, and CART cells can be continuously amplified in vivo; thirdly, because CAR-T does not need to start tumor killing in a mode of combining TCR and MHC molecules, the CAR-T has non-MHC restriction and can directly recognize tumor surface antigens, so that the tumor recognition efficiency is high, the specificity is strong, and tumor cells with down-regulated MHC can be killed. Based on these advantages of CAR-T, CAR-T has achieved significant efficacy in the treatment of hematological tumors such as B cell acute leukemia and non-Hodgkin's lymphoma, has been approved by the FDA, and has shown primary efficacy in the treatment of solid tumors.
Gastrointestinal tract tumors are a great threat to human life, and although treatment means such as surgical treatment, radiotherapy and chemotherapy, interventional therapy and the like have certain curative effect on the tumors, the survival rate of patients is not improved obviously. Currently, CAR-T cell therapy techniques of great interest are expected to be therapeutic breaks.
Carcinoembryonic antigen (CEA) is an embryo antigen rich in polysaccharide, CEA is a broad-spectrum tumor marker, can be expressed in various tumors and can appear in the serum of various tumor patients, belongs to a tumor marker which is commonly used clinically, and is mainly used for assisting diagnosis, prognosis judgment, curative effect monitoring, tumor recurrence and the like of malignant tumors clinically. Development of a CART cell therapy for CEA may be an effective therapeutic approach to the treatment of a variety of solid tumors.
Disclosure of Invention
In order to develop an immune cell therapy against CEA, the object of the present invention is, in a first aspect, to provide a chimeric antigen receptor targeting cells expressing a carcinoembryonic antigen, the chimeric antigen receptor comprising: the amino acid sequence is shown as SEQ ID NO. 1, and the single-chain antibody of the carcinoembryonic antigen is targeted.
Preferably, the chimeric antigen receptor comprises: a leader peptide, the single chain antibody, a hinge region, a transmembrane domain and an intracellular signaling domain connected in sequence.
Preferably, the Hinge region is CD8 Hinge; the Transmembrane domain is CD8 Transmembrane; the intracellular signaling domain comprises CD28 and/or 4-1BB, and CD3 z.
Preferably, the nucleotide sequence of the leader peptide is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 3; the nucleotide sequence of the hinge region is shown as SEQ ID NO. 4, and the amino acid sequence is shown as SEQ ID NO. 5; the nucleotide sequence of the transmembrane structural domain is shown as SEQ ID NO. 6, and the amino acid sequence is shown as SEQ ID NO. 7.
Preferably, the nucleotide sequence of the CD28 is shown as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO. 9; the 4-1BB nucleotide sequence is shown as SEQ ID NO. 10, and the amino acid sequence is shown as SEQ ID NO. 11; the nucleotide sequence of the CD3z is shown as SEQ ID NO. 12, and the amino acid sequence is shown as SEQ ID NO. 13.
In a second aspect of the invention, there is provided a nucleic acid molecule encoding a chimeric antigen receptor as described above which is targeted to a cell expressing a carcinoembryonic antigen.
In a third aspect of the invention, there is provided a recombinant expression vector for expressing the chimeric antigen receptor targeted to cells expressing carcinoembryonic antigen as described above.
In a fourth aspect, the invention provides an immune cell prepared using the chimeric antigen receptor described above that targets a cell expressing a carcinoembryonic antigen.
In a fifth aspect of the invention, there is provided a single chain antibody targeting carcinoembryonic antigen, said single chain antibody comprising: a heavy chain variable region and a light chain variable region; the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 14 is shown in the figure; the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 15 is shown in the figure; the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 16 is shown in the figure; the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17 is shown; the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 18 is shown in the figure; the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 19, respectively.
Preferably, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 1.
Has the advantages that:
according to the characteristics of the gastrointestinal tumor specificity high expression CEA tumor antigen, the invention designs the specificity antibody for targeting recognition CEA, can adopt the second generation or the third generation CAR skeleton as a vector, designs the CAR-T cell for targeting carcinoembryonic antigen, can effectively kill the carcinoembryonic antigen positive tumor cell, and can be used for the targeted therapy of tumor.
Drawings
FIG. 1 is a diagram showing an example of 3 structures of the chimeric antigen receptor of the present invention.
FIG. 2 is a schematic structural diagram of a lentiviral expression vector used in an embodiment of the present invention.
FIG. 3 shows the restriction enzyme XbaI cleavage electrophoresis of the lentiviral expression vector into which a nucleic acid fragment encoding a chimeric antigen receptor targeting CEA was inserted.
FIG. 4 shows the killing of CEA-CART cells and control T cells and positive control cells against carcinoembryonic antigen positive K562 tumor cells.
FIG. 5 is a graph of CEA-CART cells and control T cells and positive control cells killing of carcinoembryonic antigen negative K562 tumor cells.
FIG. 6 is a graph showing the results of IL-2 secretion by CEA-CART cells and control T cells as well as positive control cells.
FIG. 7 is a graph showing the results of secretion of IFN-gamma by CEA-CART cells and control T cells and positive control cells.
Detailed Description
The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The structure of the chimeric antigen receptor of the present invention is shown in FIG. 1. The costimulatory molecule can be selected from 4-1BB and/or CD28, and CD3z, and other immune costimulatory molecules.
The immunoreceptor tyrosine activation motif may be the CD3 ζ, fcepsilon RI γ signaling chain, preferably, the signaling domain is the CD3z signaling chain.
FIG. 2 is a schematic diagram of the structure of a lentiviral expression vector incorporating a nucleic acid fragment encoding a chimeric antigen receptor targeting carcinoembryonic antigen (CEA) with a human EF1alpha promoter, in an embodiment of the invention.
scFv refers to single chain antibodies that target carcinoembryonic antigen.
CEA-CART cells represent CAR-T cells that target carcinoembryonic antigens.
The lentiviral expression vector of the chimeric antigen receptor of the invention can be used for preparing immune cells, and the immune cells comprise T cells, B lymphocytes, NK lymphocytes, CIK cells, monocytes or neutrophils. Tumors expressing the CEA antigen include lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestine adenocarcinoma, oral cancer or nasopharyngeal carcinoma.
The method for introducing the vector into the immune cell may be a particle gun method, an electrotransfer method, a virus transfer method, and the introduction method is a virus transfer method in the examples of the present invention.
Examples
1. Construction of chimeric antigen receptor expression plasmids containing scFv targeting carcinoembryonic antigen (CEA)
A1461 bp DNA fragment shown in SEQ ID NO 20 was artificially synthesized, wherein the nucleotides at positions 1 to 63 encoded a leader, the nucleotides at positions 64 to 789 encoded a CEAscFv (scFv targeting carcinoembryonic antigen), the nucleotides at positions 790 and 924 encoded a CD8 hinge region, and the nucleotides at positions 925 and 996 encoded a transmembrane region: CD8Transmembrane (CD8 TM), the 997-position 1122 nucleotide codes for 4-1BB, and the 1123-position 1458 nucleotide codes for CD3 z. The amino acid sequence encoded by the DNA fragment shown in SEQ ID NO. 20 is shown in SEQ ID NO. 21.
The DNA fragment is inserted into the downstream of an EF1alpha promoter of a lentiviral expression vector to obtain a chimeric antigen receptor expression plasmid (pLV-CEA-scfv-BBZ) of the targeting CEA. FIG. 3 shows the restriction enzyme XbaI cleavage electrophoresis identification chart of the lentiviral expression vector inserted with the chimeric antigen receptor targeting CEA.
2. CEA-targeted chimeric antigen receptor expression plasmid transfected T cells
(1) Packaging preparation of lentiviruses
The chimeric antigen receptor expression plasmid, the structural plasmid and the envelope plasmid were transfected into 293T/17 cells by calcium phosphate transfer method at a ratio of 3:2: 1. 12 hours after transfection, fresh DMEM medium containing 10% FBS was replaced while sodium butyrate was added at a final concentration of 5 mM. 48 hours after transfection, the virus-containing cell culture supernatant was aspirated into a centrifuge tube, centrifuged at 1500rpm at 4 ℃ for 10min, transferred to a new centrifuge tube, filtered through a 0.45 μm filter, and stored at-80 ℃.
(2) Preparation of T cells
10ml of fresh blood of healthy people is taken, and peripheral blood mononuclear cells are separated by lymphocyte separation liquid (rhyme-Fei organisms), and the specific method is shown in the specification. Cell density was adjusted to 2x10 with T551 Medium containing 4% autologous serum6Adding IL-2 with the final concentration of 300U/ml and CD3 monoclonal antibody with the final concentration of 100ng/ml for induction culture for 24h to obtain T cells.
(3) Lentiviral infected T cells and expansion culture of infected T cells
Adding the slow virus solution at MOI 10 to a solution containing 2 × 1061 well of 6-well plate of the above-described T cells cultured under induction at 37 ℃ with 5% CO2Co-culturing in an incubator. After three days, the cells were washed by centrifugation, fresh T551 medium containing 300IU/ml IL-2, 100ng/ml CD3 mab and 4% human autologous serum was added, and cell density was adjusted to 2X106The culture was continued at a concentration of/ml. Cell density was measured every 2-3 days, centrifuged and adjusted with fresh medium for continuous expansion. This is repeated until the cells are expanded to a sufficient amount.
After testing for efficient expression of the chimeric antigen receptor in T cells, CAR-T cells against CEA were obtained.
Specific killing Activity of CAR-T cells against CEA-Positive malignant cells
Take 5X 104A plurality of CEA-highly expressing K562 cells (CEA positive, expressed as CEA-K562) and a control K562 cell (CEA negative, expressed as K562) were seeded into a 96-well plate, and Effector cells (Effector): CAR-T cells (CEA-CART), CAR-T positive control cells (CEA-CART positive control), and unmodified T cells (T) were added at three ratios of Target cells (Target) 10:1, 3:1, 1: 1. After incubation for 18h, the specific killing activity of CAR-T cells on K562 cells highly expressing CEA was tested according to the procedures of LDH cytotoxicity assay kit (Biyun day). The results show that the CAR-T cell group has stronger killing effect on K562 cells highly expressing CEA compared with the CAR-T positive control cell group and the unmodified T cell group, and the difference is significant (p)<0.05) (fig. 4); toThere was no significant difference in killing of K562 cells expressing CEA (p)>0.05) (fig. 5).
Among them, scFv of CAR-T positive control cells was constructed in accordance with published patent document CN 201510304389.3.
ELISA for detecting the expression of IFN-gamma, IL-2.
Take 5X 104A plurality of K562 cells (CEA positive) with high CEA expression and a control K562 cell (CEA negative) are inoculated on a 96-well plate, and Effector cells (Effector) are respectively added into two cell culture wells: CAR-T cells (CEA-CART), CAR-T positive control cells (CEA-CART positive control), and unmodified T cells (T) were added at a ratio of 3:1 for the Target cells (Target). After incubation for 18h, the supernatant was removed and assayed for IL-2 and IFN-. gamma.content following the Human IL-2ELISA Kit (Dake). As shown in FIGS. 6 and 7, the results showed that the CAR-T cell group produced a large amount of IL-2 and IFN- γ to CEA-highly expressing K562 cells at an effective target ratio of 3:1 compared to the CAR-T positive control cell group and the T cell group, and that this difference was significant (p is significant) (p is a positive control cell group and T cell group)<0.05); k562 cells that do not express CEA do not induce cytokines and there is no significant difference (p)>0.05). Therefore, the CAR-T cells have specific cytokine-induced effects on CEA-highly expressing tumor cells.
In conclusion, the chimeric antigen receptor targeting the carcinoembryonic antigen can effectively and specifically target malignant cells expressing the CEA surface antigen, thereby providing a more efficient method for treating some tumors expressing the CEA surface antigen and having fewer side effects and adverse reactions.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Sequence listing
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<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 13
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 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
<210> 14
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr
1 5 10
<210> 15
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Lys Val Ser
1
<210> 16
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Ser Gln Thr Thr His Val Pro Phe Thr
1 5
<210> 17
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Gly Phe Ser Leu Thr Thr Ser Asp Leu Gly
1 5 10
<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Ile Tyr Trp Asp Asp Asp Lys
1 5
<210> 19
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Val Arg Asn Tyr Asp Tyr
1 5
<210> 20
<211> 1461
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccgcaagtca agctggggga gtctggccct gggacattgc agccctccca gaccctcagt 120
ctgacttgtt ctttttctgg gttttcactg accacttctg acttgggtgt gagctggatt 180
cgtcagcctt caggaaaggg tctggagtgg ctggcacaca tttactggga tgatgacaag 240
tactataacc catccctgaa gagccggctc agaatctcca aagatatatc cagcaaccag 300
gttttcctca agatcgccag tgttgacatt gtagatactg ccacttacta ctgtgttcga 360
aactatgatt attggggtca aggaacctca gtcaccgtct cctcaggtgg aggaggcagc 420
ggcggtggag ggtctggtgg aggtggttct gatgttgtga tgacccaaac tccactctcc 480
ctgcctgtca gtcttggaga tcaagcctcc atctcttgca gatctagtca gagccttgta 540
cacagtaatg gaaacaccta tttacattgg tacctgcaga ggccaggcca gtctccaaag 600
ctcctgatct acaaagtttc cagccgattt tctggggtcc cagacaggtt cagtggcagt 660
ggatcaggga ctgatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt 720
tatttctgct ctcaaactac acatgtcccg ttcacgttcg gaggggggac caagctggaa 780
ataaaacgga ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140
agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440
caggccctgc cccctcgcta a 1461
<210> 21
<211> 486
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Lys Leu Gly Glu Ser Gly Pro Gly Thr
20 25 30
Leu Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe
35 40 45
Ser Leu Thr Thr Ser Asp Leu Gly Val Ser Trp Ile Arg Gln Pro Ser
50 55 60
Gly Lys Gly Leu Glu Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys
65 70 75 80
Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Leu Arg Ile Ser Lys Asp Ile
85 90 95
Ser Ser Asn Gln Val Phe Leu Lys Ile Ala Ser Val Asp Ile Val Asp
100 105 110
Thr Ala Thr Tyr Tyr Cys Val Arg Asn Tyr Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser
145 150 155 160
Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
165 170 175
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
180 185 190
Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Ser
195 200 205
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
210 215 220
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
225 230 235 240
Tyr Phe Cys Ser Gln Thr Thr His Val Pro Phe Thr Phe Gly Gly Gly
245 250 255
Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
340 345 350
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
Gln Ala Leu Pro Pro Arg
485
Claims (10)
1. A chimeric antigen receptor for targeting a cell expressing a carcinoembryonic antigen, the chimeric antigen receptor comprising: the amino acid sequence is shown as SEQ ID NO. 1, and the single-chain antibody of the carcinoembryonic antigen is targeted.
2. The chimeric antigen receptor targeted to a cell expressing a carcinoembryonic antigen as claimed in claim 1, wherein said chimeric antigen receptor is composed of a leader peptide, said single chain antibody, a hinge region, a transmembrane domain and an intracellular signaling domain connected in sequence.
3. The chimeric antigen receptor targeted to carcinoembryonic antigen expressing cells of claim 2, wherein said Hinge region is CD8 Hinge; the Transmembrane domain is CD8 Transmembrane; the intracellular signaling domain is CD28 and/or 4-1BB, and CD3 z.
4. The chimeric antigen receptor targeting cells expressing carcinoembryonic antigen as claimed in claim 3, wherein the leader peptide has the nucleotide sequence shown in SEQ ID NO. 2 and the amino acid sequence shown in SEQ ID NO. 3; the nucleotide sequence of the hinge region is shown as SEQ ID NO. 4, and the amino acid sequence is shown as SEQ ID NO. 5; the nucleotide sequence of the transmembrane structural domain is shown as SEQ ID NO. 6, and the amino acid sequence is shown as SEQ ID NO. 7.
5. The chimeric antigen receptor targeting carcinoembryonic antigen expressing cells according to claim 3, wherein the nucleotide sequence of CD28 is shown in SEQ ID NO 8 and the amino acid sequence is shown in SEQ ID NO 9; the 4-1BB nucleotide sequence is shown as SEQ ID NO. 10, and the amino acid sequence is shown as SEQ ID NO. 11; the nucleotide sequence of the CD3z is shown as SEQ ID NO. 12, and the amino acid sequence is shown as SEQ ID NO. 13.
6. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-5 targeted to a cell expressing a carcinoembryonic antigen.
7. A recombinant expression vector expressing the chimeric antigen receptor targeted to a cell expressing a carcinoembryonic antigen of any one of claims 1 to 5.
8. An immune cell expressing a chimeric antigen receptor of any one of claims 1 to 5 targeted to a cell expressing a carcinoembryonic antigen.
9. A single chain antibody targeting carcinoembryonic antigen, said single chain antibody comprising: a heavy chain variable region and a light chain variable region;
the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 14 is shown in the figure; the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 15 is shown in the figure; the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 16 is shown in the figure;
the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17 is shown; the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 18 is shown in the figure; the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 19, respectively.
10. The single chain antibody targeting carcinoembryonic antigen of claim 9, wherein the amino acid sequence of the single chain antibody is represented by SEQ ID NO. 1.
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| CN113336848B (en) * | 2021-02-03 | 2022-08-19 | 上海莱馥医疗科技有限公司 | anti-PD-1 antibody and application thereof |
Citations (3)
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|---|---|---|---|---|
| CN104910279A (en) * | 2015-06-05 | 2015-09-16 | 重庆倍思益生物科技有限公司 | Chimeric antigen receptor of targeted carcino-embryonic antigens, lentiviral expression vector as well as preparation methods and applications thereof |
| WO2018012895A1 (en) * | 2016-07-14 | 2018-01-18 | 주식회사 큐로셀 | Immune cell surmounting immune checkpoint and pharmaceutical composition containing same immune cell |
| CN110862456A (en) * | 2019-09-23 | 2020-03-06 | 华道(上海)生物医药有限公司 | Anti-carcinoembryonic antigen antibody and preparation method and application thereof |
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| CN104910279A (en) * | 2015-06-05 | 2015-09-16 | 重庆倍思益生物科技有限公司 | Chimeric antigen receptor of targeted carcino-embryonic antigens, lentiviral expression vector as well as preparation methods and applications thereof |
| WO2018012895A1 (en) * | 2016-07-14 | 2018-01-18 | 주식회사 큐로셀 | Immune cell surmounting immune checkpoint and pharmaceutical composition containing same immune cell |
| CN110862456A (en) * | 2019-09-23 | 2020-03-06 | 华道(上海)生物医药有限公司 | Anti-carcinoembryonic antigen antibody and preparation method and application thereof |
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