Nucleic acids encoding chimeric antigen receptor proteins which bind epidermal growth factor receptor and T lymphocyte expressing the protein

Abstract

A nucleic acid for coding a chimeric antigen receptor protein expressed on the surface of a human T lymphocyte. The chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain that are orderly connected. The extracellular binding domain comprises a single-chain antibody scFv (EGFR) for specific recognition of 287.sup.th to 302.sup.nd amino acid epitopes of a human epidermal growth factor receptor (EGFR).

Claims

1. A nucleic acid encoding a chimeric antigen receptor protein expressed on a surface of a human T lymphocyte, wherein the chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain, wherein the extracellular binding domain comprises a single chain antibody having at least 95% sequence homology to any one of SEQ 31-34 in the framework region, and wherein the extracellular binding domain binds to the 287.sup.th-302.sup.nd amino acid epitopes of human epidermal growth factor receptor (EGFR).

2. The nucleic acid of claim 1, wherein the intracellular signal domain is selected from the sequence of intracellular signal domain of CD3, FcRI, CD28, CD137, CD134, or combinations thereof.

3. The nucleic acid of claim 2, wherein the chimeric antigen receptor protein is the following chimeric antigen receptor protein comprising any one of: extracellular domain scFv(EGFR)-transmembrane domain CD8-intracellular domain CD3, extracellular domain scFv(EGFR)-transmembrane domain CD8-intracellular domain CD137-intracellular domain CD3, extracellular domain scFv(EGFR)-transmembrane domain CD28-intracellular domain CD28-intracellular domain CD3, extracellular domain scFv(EGFR)-transmembrane domain CD28-intracellular domain CD28-intracellular domain CD137-intracellular domain CD3, or combinations thereof.

4. The nucleic acid of claim 1, wherein the nucleic acid comprises SEQ ID NO:32.

5. The nucleic acid of claim 1, wherein the nucleic acid has a sequence of SEQ ID NO: 2.

6. The nucleic acid of claim 1, wherein the nucleic acid comprises SEQ ID NO:31.

7. The nucleic acid of claim 1, wherein the nucleic acid comprises SEQ ID NO:33.

8. The nucleic acid of claim 1, wherein the nucleic acid comprises SEQ ID NO:34.

9. The nucleic acid of claim 1, wherein the nucleic acid has a sequence of SEQ ID NO: 1.

10. The nucleic acid of claim 1, wherein the nucleic acid has a sequence of SEQ ID NO: 3.

11. The nucleic acid of claim 1, wherein the nucleic acid has a sequence of SEQ ID NO: 4.

12. The nucleic acid of claim 1, wherein the transmembrane domain comprises a hinge region, wherein the hinge region links the transmembrane domain to the extracellular binding domain.

13. The nucleic acid of claim 12, wherein the transmembrane domain comprises the sequence of transmembrane domain and hinge region of CD8 or CD28.

14. A vector comprising a nucleic acid of encoding a chimeric antigen receptor protein expressed on a surface of a human T lymphocyte, wherein the chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain, and wherein the extracellular binding domain comprises a single-chain antibody scFv having at least 95% sequence homology to any one of SEQ 31-34 in the framework region, and wherein the extracellular binding domain binds to the 287th-302nd amino acid epitopes of human epidermal growth factor receptor (EGFR).

15. The vector of claim 14, wherein the vector is derived from a lentiviral plasmid vector pPWT-eGFP.

16. A virus comprising a nucleic acid encoding a chimeric antigen receptor protein expressed on a surface of a human T lymphocyte, wherein the chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain, and wherein the extracellular binding domain comprises a single-chain antibody scFv having at least 95% sequence homology to any one of SEQ 31-34 in the framework region, and wherein the extracellular binding domain binds to the 287th-302nd amino acid epitopes of human epidermal growth factor receptor (EGFR).

17. A transgenic T lymphocyte, which is transduced with a nucleic acid of encoding a chimeric antigen receptor protein expressed on a surface of a human T lymphocyte, wherein the chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain, and wherein the extracellular binding domain comprises a single-chain antibody scFv having at least 95% sequence homology any one of SEQ 31-34 in the framework region, and wherein the extracellular binding domain binds to the 287th-302nd amino acid epitopes of human epidermal growth factor receptor (EGFR).

18. A transgenic T lymphocyte, wherein a chimeric antigen receptor is expressed on the surface of the T lymphocyte and the chimeric antigen receptor is encoded by a nucleic acid of encoding a chimeric antigen receptor protein expressed on a surface of a human T lymphocyte, wherein the chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain, and wherein the extracellular binding domain comprises a single-chain antibody scFv having at least 95% sequence homology any one of SEQ 31-34 in the framework region, and wherein the extracellular binding domain binds to the 287th-302nd amino acid epitopes of human epidermal growth factor receptor (EGFR).

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows, as an example, a schematic diagram of the structure of the lentiviral vector pWPT-eGFP-F2A-CAR of the present invention comprising CAR-encoding sequence.

(2) FIG. 2 shows, as an example, a schematic diagram of linkages between different domains of the CAR of the present invention contained in a lentiviral vector, in which eGFP and svFv (EGFR)-specific chimeric antigen receptors are linked by ribosome skipping sequence F2A.

(3) FIG. 3 shows a nucleic acid electrophoresis map of the lentiviral plasmid of Example 1 identified by double digestion of MluI and SalI. Wherein M1 is a DS2000 molecular weight marker (Guangzhou Dongsheng Biotechnology Co., Ltd.); and M2 is a Hind III marker (Guangzhou Dongsheng Biotechnology Co., Ltd.). Lanes 1-6 are 1: pWPT-eGFP; 2: pWPT-eGFP-F2A-806- Z; 3: pWPT-eGFP-F2A-806-Z; 4: pWPT-eGFP-F2A-806-BBZ; 5: pWPT-eGFP-F2A-806-28Z; 6: pWPT-eGFP-F2A-806-28BBZ, respectively.

(4) FIG. 4 shows the results of flow cytometry of eGFP expressed in CD8.sup.+ T lymphocytes infected with the virus of Example 2 of the present invention.

(5) FIG. 5 shows the in vitro growth of CD8.sup.+ T lymphocytes expressing different chimeric antigen receptors (CAR.sup.+) according to Example 2 of the present invention.

(6) FIG. 6 shows the results of flow cytometry of the expression of EGFR287-302 epitope on the surface of various tumor cell lines used in Example 3 of the present invention.

MODES FOR CARRYING OUT THE INVENTION

(7) The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples without particular conditions, they are performed under routine conditions, such as conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturer, when the specification of the reagent company is specifically mentioned in the Examples.

Example 1. Construction of Lentiviral Plasmid Expressing the Chimeric Antigen Receptor of the Present Invention

(8) The connection order of each part in the exemplary chimeric antigen receptor of the invention is illustrated in following Table 1, which is also shown in FIG. 2.

(9) TABLE-US-00001 TABLE 1 extracellular binding chimeric domain-transmembrane antigen domain-intracellular signal domain 1- receptor intracellular signal domain 2 Description 806- Z scFv(EGFR)-CD8-CD3 zeta Negative control 806-Z scFv(EGFR)-CD8-CD3 zeta The 1.sup.st generation 806-BBZ scFv(EGFR)-CD8-CD137-CD3 zeta The 2.sup.nd generation 806-28Z scFv(EGFR)-CD28-CD28-CD3 zeta The 2.sup.nd generation 806-28BBZ scFv(EGFR)-CD28-CD28-CD137-CD3 The 3.sup.rd zeta generation

(10) 1. Amplification of Nucleic Acid Fragments

(11) (1) Amplification of scFv(EGFR) Sequence

(12) The sequence of scFv (EGFR) sequence was amplified by using single-stranded bifunctional antibody nucleotide 806/CD3 or hu7B3/CD3 constructed in our laboratory as template. The sequence of the template is shown in Chinese Patent Application 201210094008.x as SEQ ID NO: 10 and 11, respectively. And the primers used in amplification are:

(13) upstream primer 5-gacatcctgatgacccaatctccatcctc-3 (SEQ ID NO: 5) and downstream primer 5-tgcagagacagtgaccagagtcccttgg-3 (SEQ ID NO: 6), used to amplify 806 scFv (EGFR);

(14) and upstream primer 5-gatattcagatgacccagagcccg-3 (SEQ ID NO: 7) and downstream primer 5-gctgctcacggtcaccagggtg-3 (SEQ ID NO: 8), used to amplify hu7B3 scFv (EGFR).

(15) In both situations, the size of amplified target band was 720 bp. PCR amplification conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 40 s; annealing at 58 C. for 40 s; extension at 68 C. for 40 s; 25 cycles; followed by a total extension at 68 C. for 10 min. PCR-amplified bands were confirmed by agarose gel electrophoresis to comply with the predicted fragment size.

(16) The sequence of negative control scFv (CD19) was determined according to the sequence of FMC63-28Z (HM852952.1) from GenBank, and the sequence was obtained by Shanghai Ruijin Biotechnology Co. through whole-gene synthesis.

(17) (2) Nucleic Acid Sequence of Other Parts of Chimeric Antigen Receptor

(18) Other parts of the chimeric antigen receptor protein and the hinge region connecting these parts were amplified as follows: 1 ml Trizol (Invitrogen) was added into 110.sup.7 healthy human peripheral blood mononuclear cells (provided by Shanghai Blood Center) for the lysis of cells; afterfwards, total RNA was extracted by phenol-chloroform method; and cDNAs were prepared through reverse transcription by using ImProm-II Reverse Transcription Kit (Promaga). Above prepared cDNAs were used as templates in:

(19) (a) amplification to obtain CD8a hinge region-transmembrane domain using upstream primer 5-cactgtctctgcaaccacgacgccagcg-3 (SEQ ID NO: 9) and downstream primer 5-ggtgataaccagtgacaggag-3 (SEQ ID NO: 10); and PCR amplification conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 30 s; annealing at 58 C. for 30 s; extension at 68 C. for 30 s; 25 cycles; followed by a total extension at 68 C. for 10 min. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 198 bp.

(20) (b) amplification to obtain CD8a hinge region-transmembrane domain-delta Z(Z) by using upstream primer 5-cactgtctctgcaaccacgacgccagcg-3 (SEQ ID NO: 11) and downstream primer 5-gaggtcgacctacgcgggggcgtctgcgctcctgctgaacttcactctggtgataaccagtg-3 (SEQ ID NO: 12); and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 234 bp.

(21) (c) amplification to obtain CD28 transmembrane domain-Intracellular signal domain fragment by using upstream primer 5-ttttgggtgctggtggtggttgg-3 (SEQ ID NO: 13) and downstream primer 5-gctgaacttcactctggagcgataggctgcgaag-3 (SEQ ID NO: 14); and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 465 bp.

(22) (d) amplification to obtain CD137 Intracellular domain by using upstream primer 5-aaacggggcagaaagaaactc-3 (SEQ ID NO: 15) and downstream primer 5-cagttcacatcctccttc-3 (SEQ ID NO: 16); and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 126 bp.

(23) (e) amplification to obtain CD3 zeta signal domain by using upstream primer 5-cactggttatcaccagagtgaagttcagcaggagc-3 (SEQ ID NO: 17) and downstream primer 5-cgaggtcgacctagcgagggggcagggcctgcatg-3 (SEQ ID NO: 18); and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 339 bp.

(24) 2. Splicing of Nucleic Acid Fragments

(25) (a) upstream primer 5-accacgacgccagcgccg-3 (SEQ ID NO: 19) and downstream primer 5-cacccagaaaataataaag-3 (SEQ ID NO: 20) were used in splicing to obtain CD8a hinge region-CD28 transmembrane domain; and splicing conditions were: pre-denaturation of CD8a hinge region (50 ng)+CD28 transmembrane domain (50 ng) at 94 C. for 4 min; denaturation at 94 C. for 30 s; annealing at 60 C. for 30 s; extension at 68 C. for 30 s; 5 cycles; followed by a total extension at 68 C. for 10 min; DNA polymerase and upstream and downstream primers were supplemented, and afterwards PCR amplification was performed for 25 cycles; and PCR amplification conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 30 s; annealing at 60 C. for 30 s; extension at 68 C. for 30 s; 25 cycles; followed by a total extension at 68 C. for 10 min. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 216 bp.

(26) (b) upstream primer 5-agagtgaagttcagcaggagcgcag-3 (SEQ ID NO:21) and downstream primer 5-cgaggtcgacctagcgagggggcagggcctgcatg-3 (SEQ ID NO:18) were used in splicing to obtain 4-1BB intracellular signal domain and CD3 zeta. i.e., BBZ; and splicing and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 465 bp.

(27) (c) upstream primer 5-cactgtctctgcaaccacgacgccagcg-3 (SEQ ID NO: 22) and downstream primer 5-cgaggtcgacctagcgagggggcagggcctgcatg-3 (SEQ ID NO: 18) were used to splice equimolar of CD8a hinge region-transmembrane domain and CD3 zeta (about 50 ng), and CD8-CD3 zeta (i.e., CD8-Z) was obtained through PCR amplification; and splicing and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 537 bp.

(28) (d) upstream primer 5-cactgtctctgcaaccacgacgccagcg-3 (SEQ ID NO: 23) and downstream primer 5-cgaggtcgacctagcgagggggcagggcctgcatg-3 (SEQ ID NO: 18) were used to splice CD8 hinge region-transmembrane domain and BBZ, and the target fragment, CD8-CD137-CD3 zeta (i.e., CD8-BBZ) was obtained through PCR amplification; and splicing and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 663 bp.

(29) (e) upstream primer 5-accacgacgccagcgccg-3 (SEQ ID NO: 24) and downstream primer 5-cgaggtcgacctagcgagggggcagggcctgcatg-3 (SEQ ID NO: 18) were used to splice CD8a hinge region-CD28 transmembrane domain and Z, and the target fragment, CD8 hinge region-CD28 transmembrane domain-28 Z intracellular domain was obtained through PCR amplification; and splicing and PCR amplification conditions were the same as above. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 678 bp.

(30) (f) upstream primer 5-accacgacgccagcgccg-3 (SEQ ID NO: 19) and downstream primer 5-cgaggtcgacctagcgagggggcagggcctgcatg-3 (SEQ ID NO: 18) were used to splice CD8 hinge region, CD28 transmembrane domain-intracellular signal domain fragment obtained through PCR and BBZ, to obtain the target fragment, CD8 hinge region-CD28TM-28BBZ. The spliced and amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 804 bp.

(31) (g) Z, Z and 28BBZ nucleic acid fragments comprising hinge region and the transmembrane region respectively obtained by equimolar amplification were spliced with equimolar single-chain antibody nucleic acid sequence scFv806 or scFvCD19 (about 50 ng), and amplified by PCR, nucleic acid sequences encoding chimeric antibodies 806-Z, 806-Z, 806-BBZ, 806-28Z and 806-28BBZ were obtained by splicing in the pattern shown in FIG. 2, and splicing and PCR amplification conditions were the same as above.

(32) 3. Construction of Plasmid Vector

(33) The vector system used in this example belongs to the third-generation of self-inactivating lentiviral vector system. The system consists of three plasmids, namely packaging plasmid psPAX2 encoding Gag/Pol protein, Rev protein; envelope plasmid PMD2.G encoding VSV-G protein; and recombinant expression vector encoding target gene CAR based on blank vector pPWT-eGFP.

(34) In the blank vector pPT-eGFP, the expression of enhanced green fluorescent protein (eGFP) was regulated by the promoter of elongation factor-1 (EF-1). And in the recombinant expression vector encoding target gene CAR, the co-expression of eGFP and the target gene CAR was achieved by a ribosome jumping sequence 2A from foot-and-mouth disease virus (FMDV, F2A). F2A is a core sequence of 2A (or self-cleaving polypeptide 2A) from foot-and-mouth disease virus, which possesses self-cleaving function of 2A and enables co-expression of upstream and downstream genes. 2A provides an effective and feasible strategy for constructing multicistronic vectors of gene therapy due to advantages of high cleaving efficiency, high balance of expression of upstream and downstream gene and short sequence. Especially in the immunotherapy of chimeric antigen receptor gene-modified T lymphocytes, the co-expression of target gene and GFP or eGFP is generally achieved by using such sequence, and the expression of CAR can be indirectly detected by detecting GFP or eGFP.

(35) In this example, a lentiviral expression vector co-expressing eGFP and specific CAR linked by F2A was constructed, collectively named as pWPT-eGFP-F2A-CAR. Steps for splicing each part of eGFP-F2A-CAR are as follows:

(36) Fragment of F2A (66 bp)-CD8a signal peptide (63 bp) and a small nucleic acid sequence (about 18 bp) fused with upstream eGFP and downstream CAR was obtained by primer splicing, the theoretical size of which was 165 bp. And the primers were:

(37) TABLE-US-00002 (SEQIDNO:25) 5-attcaaagtctgtttcacgctactagctagtccg-3 (SEQIDNO:26) 5-gtgaaacagactttgaattttgaccttctgaagttggcaggagacgt tgagtccaac-3 (SEQIDNO:27) 5-agcggcaggagcaaggcggtcactggtaaggccatgggcccagggtt ggactcaacgtc-3 (SEQIDNO:28) 5-ctcctgccgctggccttgctgctccacgccgccaggccggacatcct gatgacccaatc-3

(38) Splicing conditions for primer were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 20 s; annealing at 50 C. for 20 s; extension at 68 C. for 30 s; 25 cycles; followed by a total extension at 68 C. for 10 min. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size.

(39) Upstream primer 5-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 (SEQ ID NO:29) and downstream primer 5-gctactagctagtccggacttgtacagctcgtccatg-3 (SEQ ID NO:30) were used to amplify target gene eGFP, by using pWPT-eGFP blank vector as template. PCR amplification conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 40 s; annealing at 56 C. for 40 s; extension at 68 C. for 40 s; 25 cycles; followed by a total extension at 68 C. for 10 min. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 735 bp.

(40) Upstream primer 5-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 (SEQ ID NO: 29) and downstream primer 5-gaggtcgacctacgcgggggcgtctgcgctcctgctgaacttcactctggtgataaccagtg-3 (SEQ ID NO: 12) were used to splice equimolar of above obtained F2A-CD8 signal peptide fragment, eGFP and 806- Z (about 80 ng) to obtain eGFP-F2A-806- Z. And the splicing conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 40 s; annealing at 62 C. for 40 s; extension at 68 C. for 140 s; 5 cycles; afterwards, DNA polymerase and upstream and downstream primers in a suitable volume were supplemented, and PCR amplification was performed for 25 cycles; and PCR amplification conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 30 s; annealing at 62 C. for 40 s; extension at 68 C. for 140 s. The amplified product was confirmed by agarose gel electrophoresis to comply with the theoretical size, 1861 bp.

(41) Upstream primer 5-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 (SEQ ID NO: 29) and downstream primer 5-gaggtcgacctagcgagggggcagggcctgcatgtgaag-3 (SEQ ID NO: 18) were used to splice equimolar of above obtained F2A and CD8a signal peptide fragments, eGFP and 806-Z, 806-BBZ, CD19-BBZ, 806-28Z as well as 806-28BBZ (about 80 ng) respectively. And the splicing conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 40 s; annealing at 62 C. for 40 s; extension at 68 C. for 140 s; 5 cycles; afterwards, DNA polymerase and upstream and downstream primers in a suitable volume were supplemented, and PCR amplification was performed for 25 cycles; and PCR amplification conditions were: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 40 s; annealing at 62 C. for 40 s; extension at 68 C. for 140 s.

(42) eGFP-F2A-806-Z, eGFP-F2A-806-BBZ, eGFP-F2A-806-28Z and eGFP-F2A-806-28BB Z were obtained, the theoretical size of which were 2164 bp, 2290 bp, 2305 bp, 2431 bp respectively, and the amplified products were confirmed by agarose gel electrophoresis to comply with the theoretical sizes, wherein MluI and SalI cleavage sites were introduced upstream and downstream of the open reading frame. The above obtained target gene eGFP-F2A-CAR was digested with MluI and SalI, and ligated into pWPT vector digested with the same enzymes. The constructed lentiviral vector expressing each chimeric antigen receptor was digested by MluI and SalI (FIG. 3) and the sequence was determined as correct for lentivirus packaging.

(43) As described previously, eGFP-F2A-CAR was transcribed into mRNA, but translated into two proteins, eGFP and anti-EGFR287-302 chimeric antigen receptor. Under the action of CD8 signal peptide, anti-EGFR287-302 chimeric antigen receptor will be located on cell membrane.

(44) 4. 293Tcells were Transfected by Plasmids for Packaging Lentivirus

(45) 293T cells (ATCC: CRL-11268) which were cultured to 6.sup.th-10.sup.th generation were inoculated into a 10 cm dish at a density of 610.sup.6, and cultured overnight at 37 C., 5% CO.sub.2 for transfection. The medium was DMEM (PAA) containing 10% fetal bovine serum (PAA). The next day, the medium was replaced with serum-free DMEM at about 2 hours before transfection.

(46) The procedure for transfection was as follows

(47) 4.1 20 g of blank plasmid pWPT-GFP (mock control) or 20 g of target gene plasmid pWPT-eGFP-F2A-CAR were mixed with 15 g of packaging plasmid PAX2 and 6 g of envelope plasmid pMD2.G into 500 L of MillQ water,

(48) 4.2 62 L of 2.5 M CaCl.sub.2 (Sigma) was added dropwise and mixed at 1200 rpm/min vortex,

(49) 4.3 Finally, 500 L of 2HeBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4.2H2O, 12 mM glucose, 50 mM Hepes, Sigma, pH 7.0, 0.22 M) was added dropwise and was mixed at 1200 rpm/min vortex for 10 seconds,

(50) 4.4 immediately added dropwise to the Petri dish, gently shaken, cultured at 37 C., 5% CO.sub.2 for 46 h, and replaced with DMEM containing 10% fetal calf serum.

(51) Transfection efficiency was observed on the next day of transfection (i.e., the proportion of cells with green fluorescence), and 80% of positive transfection efficiency was deemed as successful transfection. After 48 h or 72 h of transfection, virus was collected by filtration using a 0.45 m filter (Millipore) and centrifuged at 28,000 rpm for 2 hours at 4 C. using Beckman Optima L-100XP ultracentrifuge. The supernatant was discarded, and the obtained pellet was resuspended in Quantum 007 culture liquid (PAA) at 1/10 1/50 volume of stock solution, and stored at 80 C. at 100 L/tube for virus titration or infection of T lymphocytes.

(52) 5. Determination of Titer of Lentivirus with Packaged Mock or GFP-F2A-CAR

(53) On the first day, 293T cells were inoculated at 110.sup.5/mL into 96-well culture plate, 100 L/well, and cultured at 37 C., 5% CO.sub.2, and the culture medium was DMEM containing 10% fetal bovine serum. The next day, 50 L/well of culture supernatant was discarded, 50 L/well of the fresh culture medium was added, polybrene at a final concentration of 6 g/mL was contained, and cells were cultured for 30 minutes at 37 C. and 5% CO.sub.2. 10 L/well of virus stock solution or 1 L/well of virus concentrate was added at 5-fold dilution, and 4 gradients, in duplicate, and cells were cultured at 37 C., 5% CO.sub.2. 48 h after infection, eGFP was detected by flow cytometry, the number of cells at the positive rate of 5 to 20% was appropriate, and titer (U/mL) was calculated according to positive ratedilution multiple100104. The titer of packaged virus through calcium phosphate transfection method was about 0.5210.sup.6 U/mL, and after concentration, the titer of virus was detected as about 210.sup.7 U/mL.

Example 2. Infection of CD8+ T Lymphocytes by Recombinant Lentivirus

(54) Human peripheral blood mononuclear cells were obtained from healthy human peripheral blood (provided by Shanghai Blood Center) by density gradient centrifugation. CD8.sup.+ T lymphocytes were obtained from peripheral blood mononuclear cells through negative sorting method by using CD8.sup.+ T lymphocyte beads (Stem Cell Technologies). Sorted CD8.sup.+ T lymphocytes were tested for the purity of CD8.sup.+ T lymphocytes through flow Cytometry, and if the positive rate of CD8.sup.+ T lymphocyte is 95%, it is appropriate for the next operation. Quantum 007 lymphocyte culture medium (PAA) was added at a density of about 110.sup.6/mL for culture, magnetic beads coated with anti-CD3 and CD28 antibodies (Invitrogen) were added at cell: magnetic bead of 1:1, and cells were stimulated and cultured for 24 h with recombinant human IL-2 (Shanghai Huaxin Biotechnology Co., Ltd.) at a final concentration of 100 U/mL. And then, CD8.sup.+ T lymphocytes were infected by the above recombinant lentivirus at MOI 5. Infected cells were passaged every other day at a density of 510.sup.5/mL and recombinant human IL-2 was supplemented in the lymphocyte culture medium at a final concentration of 100 U/mL.

(55) At the 7.sup.th day of culture, infected CD8.sup.+ T lymphocytes were detected by flow cytometry for the expression of different chimeric antigen receptors, detected eGFP-positive cells were deemed as positive cells expressing chimeric antigen receptors due to the co-expression of eGFP and CAR (FIG. 4). Positive ratio of CD8.sup.+ T lymphocytes infected by the virus and expressing different chimeric antigen receptors are shown in the following table, with uninfected T lymphocytes as negative control. The positive rate demonstrates that certain positive rate of CAR.sup.+ T lymphocytes can be obtained by lentivirus infection.

(56) TABLE-US-00003 TABLE 2 CD8.sup.+ T lymphocytes transfected eGFP positive rate of CD8.sup.+ T with the following CARs lymphocyte 806- Z 38.8% 806-Z 62% 806-BBZ 45% 806-28Z 78% 806-28BBZ 33.8%

(57) After infected by viruses packaging different chimeric antigen receptors, CD8.sup.+ T lymphocytes were subcultured at a cell density of 510.sup.5/ml every other day and counted. And IL-2 (final concentration of 100 U/ml) was supplemented into cell culture fluid for subculture. On the 14.sup.th day of culture, about 3555 folds of amplification can be observed (see FIG. 5), indicating that CD8.sup.+ T lymphocytes expressing different chimeric antigen receptors could be amplified in vitro in a certain quantity, thereby guaranteeing subsequent in vitro toxicity test and in vivo test.

Example 3. Detection of Exposure of EGFR287-302 Epitope in Epithelial-Derived Tumor Cell Lines

(58) Exposure of EGFR287-302 epitope on several epithelial-derived tumor cells was examined by flow cytometry using a fluorescence activated cell sorter (FACSCalibur, Becton Dickinson). Used materials include:

(59) (1) Monoclonal antibody CH12, which was constructed by our laboratory and can recognize this site (see CN 101602808B, Examples 1-4 for the construction method) was used as primary antibody (final concentration 20 g/ml, 100 L/sample),

(60) (2) FITC-labeled goat anti-human IgG was used as secondary antibody (AOGMA).

(61) The detection method for the exposure of epitope is as follows:

(62) 1. Tumor cells as shown in Table 3 in logarithmic growth phase were inoculated into 6 cm dishes at a cell density of about 90%, and incubated overnight at 37 C.

(63) 2. The cells were digested with 10 mM EDTA, collected by centrifugation at 200 g5 min, resuspended in phosphate buffer (1% NBS/PBS) containing 1% fetal bovine serum at a concentration of 110.sup.7/mL and added to a tube for flow cytometry at 100 l/tube.

(64) 3. The cells were centrifuged at 200 g5 min, and the supernatant was discard.

(65) 4. In the experimental group, CH12 was added, while in the control group, irrelevant antibody was added as the negative control. In another control group, PBS without antibody was added as blank control. The final concentration of each antibody was 20 g/ml, and 100 ul was added into each tube, and placed into an ice bath for 45 minutes.

(66) 5. 2 ml of 1% NBS/PBS was added into each tube, and centrifuged at 200 g5 min for two times.

(67) 6. The supernatant was discarded, and 1:50 dilution of FITC-labeled goat anti-human IgG was added and placed into an ice bath for 45 minutes.

(68) 7. 2 ml of 1% NBS/PBS was added into each tube, and centrifuged at 200 g5 min for two times.

(69) 8. The supernatant was discarded, and cells were resuspended in 300 ul of 1% NBS PBS and detected by flow cytometry.

(70) 9. Data were analyzed using WinMDI 2.9, a data analysis software of flow cytometry.

(71) Results are shown in FIG. 6, wherein EGFR287-302 epitope was not detected in glioma cell line U87, while in U87-EGFR over-expressing exogenous EGFR (constructed and preserved in our laboratory, and the construction method is based on Wang H., et al., Identification of an Exon 4-Deletion Variant of Epidermal Growth Factor Receptor with Increased Metastasis-Promoting Capacity. Neoplasia, 2011, 13, 461-471) and U87-EGFRvIII with over-expressing EGFRvIII (constructed and preserved in our laboratory, and the construction method is based on WO/2011/035465), EGFR287-302 epitope was detected. Additionally, exposure of EGFR287-302 epitope can be detected in three pancreatic cancer cell lines PANC-I, CFPAC-I and BxPC-3.

(72) TABLE-US-00004 TABLE 3 Name of cell Source Characteristic U87 ATCC HTB-14 Low-expression of EGFR U87- constructed and preserved in Over-expression of EGFRvIII our laboratory EGFRvIII U87-EGFR constructed and preserved in Over-expression our laboratory of EGFR PANC-1 ATCC CRL-1469 Over-expression of EGFR CFPAC-1 ATCC CRL-1918 Over-expression of EGFR BxPC-3 ATCC CRL-1687 Over-expression of EGFR

Example 4. In Vitro Experiment on Toxic Efficacy of Cells Expressing Chimeric Antigen Receptor

(73) Materials used in the in vitro toxicity experiment are as follows:

(74) Target cells were 6 types of cells as shown in the above table. Effector cells were positive cells which were in vitro cultured for 12 days and detected as expressing chimeric antigen receptor by FASC, which were marked as chimeric antigen receptor positive (CAR.sup.+) CD8.sup.+ T lymphocytes.

(75) The effector target rates were 3:1, 1:1 and 1:3 or 5:1, 2.5:1 and 1:1, respectively. The number of target cells was 10000/well, and the number of effector cells corresponded to different effector target rates. In each group, four replicates were set, and the average of the four replicate wells was taken. Detection time was 18 h or 20 h.

(76) Wherein each experiment group and each control group were as follows:

(77) each experiment group: each target+CD8.sup.+ T lymphocytes expressing different chimeric antigen receptors,

(78) Control group 1: maximum release of LDH from target cells,

(79) Control group 2: spontaneous release of LDH from target cells,

(80) Control group 3: spontaneous release of LDH from effector cells.

(81) Detection method: CytoTox 96 non-radioactive cytotoxicity assay kit (Promega) was used to perform the method. The method is a detection method based on colorimetric method, which can replace 51Cr release method. CytoTox 96 assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme which is released during cell lysis and is released in the same manner as 51Cr released in radioactivity analysis. The culture supernatant, in which LDH is released, can be detected through enzymatic reaction of 30-minute conjugation, in which LDH converts a tetrazolium salt (INT) into a red formazan. The amount of formed red product is directly proportional to the number of lysed cells. Details can be found in CytoTox 96 non-radioactive cytotoxicity test kit instructions.

(82) The formula for calculating cytotoxicity was:

(83) $\mathrm{Cytotoxicity}\%=\frac{\mathrm{Experiment}\mathrm{group}\text{-}\mathrm{control}\mathrm{group}2\text{-}\mathrm{control}\mathrm{group}3}{\mathrm{Control}\mathrm{group}1\text{-}\mathrm{control}\mathrm{group}2}100\%$

(84) Experiment results demonstrate that:

(85) The CD8.sup.+ T lymphocytes expressing scFv (EGFR)-806-Z CAR.sup.P and CD8.sup.+ T lymphocytes expressing 806-28BBZ CAR.sup.+ of the present invention showed very significant cytotoxicity against tumor cells U87-EGFRvIII, which were 55.5% and 85%, respectively.

(86) Additionally, the cytotoxicity of CD8.sup.+ T lymphocytes of the present invention described above is highly tumor-specific, since the CD8.sup.+ T lymphocytes showed high cytotoxicity against tumor cells U87-EGFRvIII with the exposure of EGFR287-302 epitope, while showed low cytotoxicity against tumor cells U87 without the exposure of EGFR287-302 epitope, less than 2% in both cases. At the same time, the mock-transfected T cells used as blank control of the evidence of experiment reliability and chimeric antigen receptor 806-Z transgenic T cells, as a negative control for evaluating effects of effector molecules in primitive T cells, showed similar low cytotoxicity against U87 and U87-EGFRvIII. The above experiment was performed at a effector to target rate of 5:1 and an action time of 20 h.

(87) TABLE-US-00005 TABLE 4 Cytotoxicity against target cells % CAR.sup.+ T cells U87 U87-EGFRvIII 806-Z CAR.sup.+ <2 56 806-28BBZ CAR.sup.+ <2 85 806- Z CAR.sup.+ <2 5 mock CAR.sup.+ <2 8

(88) In addition, under different effector to target rates, the cytotoxicity of CD8.sup.+ T lymphocytes expressing scFv (EGFR)-806-Z CAR.sup.+ and CD8.sup.+ T lymphocytes expressing 806-28BBZ CAR.sup.+ of the present invention against tumor cells U87-EGFR and U87-EGFRvIII as well as pancreatic cancer cell lines PANC-1, CFPAC-1 and BxPC-3 exhibited dependence on effector to target rate gradient. As shown in the following table, the higher the effector to target rate, the higher the cytotoxicity.

(89) TABLE-US-00006 Cytotoxicity % 806-28BBZ 806-Z CD19-BBZ at different at different at different effector to effector to effector to target rates target rates target rates 3:1 1:1 1:3 3:1 1:1 1:3 3:1 1:1 1:3 U87 2 14 25 n/a n/a n/a n/a n/a n/a U87-EGFR 98 49 29 n/a n/a n/a n/a n/a n/a U87-EGFRvIII 81 41 13 n/a n/a n/a n/a n/a n/a PANC-1 65 28 13 60 34 18 7 11 8 CFPAC-1 40 22 9 43 35 12 8 8 6 BxPC-3 70 49 24 44 24 14 7 10 8

(90) When the effector to target rate was 3:1, the cytotoxicity of CD8.sup.+ T lymphocytes expressing 806-28BBZ CAR.sup.+ against U87-EGFR was up to 98%, the cytotoxicity against U87-EGFRvIII was up to 81%, and the cytotoxicity against pancreatic cancer cell lines PANC-1, CFPAC-1 and BxPC-3 was 65%, 40% and 70% respectively.

(91) However, the cytotoxicity of CD8.sup.+ T lymphocytes expressing chimeric antigen receptor CD19-BBZ CAR.sup.+, as a negative control for evaluating effects of non-specific scFv in chimeric antigen receptor, against the above pancreatic cancer cell lines is lower than 10%, and no dependence on effector to target rate gradient was observed.