Chimeric antigen receptor and application thereof

Abstract

Provided are a chimeric antigen receptor and an application thereof; said chimeric antigen receptor contains a domain which identifies any one of, or a combination of at least two of, the malarial protein VAR2CSA, a protein tag on malarial protein VAR2CSA, or a compound capable of labeling the malarial protein VAR2CSA. The chimeric antigen receptor can identify the VAR2CSA protein or the recombinant protein (rVAR2) of any one of, or at least two of, the domains of the VAR2CSA protein which can bind to placental-like chondroitin sulfate A (pl-CSA). The VAR2CSA protein or rVAS2 protein is capable of targeting several different types of tumor cells by means of binding to the pl-CSA on the surface of the tumor cell.

Claims

1. A chimeric antigen receptor, wherein the chimeric antigen receptor contains an antigen recognition domain, wherein the antigen recognition domain is a domain which recognizes a malarial protein VAR2CSA or a recombinant protein comprising any one or at least two placental-like chondroitin sulfate A-binding domains of the malarial protein VAR2CSA, wherein the antigen recognition domain contains a variable heavy chain and a variable light chain, wherein the variable heavy chain comprises: CDR1 having an amino acid sequence as shown in SEQ ID NO. 13; CDR2 having an amino acid sequence as shown in SEQ ID NO. 14; and CDR3 having an amino acid sequence as shown in SEQ ID NO. 15; and the variable light chain comprises: CDR1 having an amino acid sequence as shown in SEQ ID NO. 16; CDR2 having an amino acid sequence as shown in SEQ ID NO. 17; and CDR3 having an amino acid sequence as shown in SEQ ID NO. 18.

2. The chimeric antigen receptor according to claim 1, wherein the antigen recognition domain recognizes an ID2a epitope of the VAR2CSA, wherein the ID2a contains an amino acid sequence as shown in SEQ ID NO. 4.

3. The chimeric antigen receptor according to claim 1, wherein the variable heavy chain contains an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence having at least 90% amino acid sequence homology to the amino acid sequence as shown in SEQ ID NO. 19; and the variable light chain contains an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having at least 90% amino acid sequence homology to the amino acid sequence as shown in SEQ ID NO. 20.

4. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further comprises any one of, or a combination of at least two of, a hinge region, a transmembrane domain and an intracellular costimulatory signal region; wherein, the hinge region is a human CD8a hinge region; the transmembrane domain is a human CD28 transmembrane domain; and the intracellular signal region is any one of, or a combination of at least two of, a human CD27 intracellular signal region, a human CD134 intracellular signal region, a human CD28 intracellular signal region, or a human 4-1BB intracellular signal region.

5. The chimeric antigen receptor according to claim 1 for transfecting and expanding CAR-T cells.

6. A cell system, comprising: an immune effector cell expressing the chimeric antigen receptor according to claim 1; and a malarial protein VAR2CSA, or a recombinant protein comprising any one or at least two placental-like chondroitin sulfate A-binding domains of the malarial protein VAR2CSA.

7. The cell system according to claim 6, wherein the immune effector cell is any one of a T cell, a B cell, an NK cell, an NKT cell, a dendritic cell or macrophage.

8. A pharmaceutical composition, comprising the cell system according to claim 6, and pharmaceutically acceptable excipients.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 (A) shows the 10% SDS-PAGE protein gel electrophoresis analysis result of the prokaryotically expressed and purified rVAR2 recombinant protein with a Strep-tag protein tag, and FIG. 1 (B) shows the 10% SDS-PAGE protein gel electrophoresis analysis result of the eukaryotically expressed and purified rVAR2 recombinant protein with a 3FLAG-tag protein tag,

(2) FIG. 2 shows detection of the function of murine anti-rVAR2 protein antibody using enzyme-linked immunosorbent assay (ELISA), wherein NC is the negative control for the ELISA detection obtained by diluting the serum of un-immunized mice at a ratio of 1:500, and the remaining groups are different dilution ratios of the antibody in the experimental group, and wherein the coating concentration of antigen (rVAR2 recombinant protein) in all control and experimental groups is 1 g/ml;

(3) FIG. 3 shows detection of the targeting specificity of rVAR2 protein to tumor cells using flow cytometry;

(4) FIG. 4 shows identification of the binding effect of monoclonal antibody 5H4 to rVAR2 protein using Western blot, where A is the prokaryotically expressed and purified rVAR2 protein, and B is the eukaryotically expressed and purified rVAR2 protein;

(5) FIG. 5 shows 12% SDS-PAGE protein gel electrophoresis detection of prokaryotically expressed and purified ID1, DBL2X and ID2a polypeptides with Strep-tag protein tags, where M is the protein molecular weight standard; NC is the negative control, that is the lysate of E. coli transfected by empty vectors; ID2a is the purified ID2a protein with Strep-tag, DBL2X is the purified DBL2X protein with Strep-tag, and ID1 is the purified ID1 protein with Strep-tag;

(6) FIG. 6 shows the epitope screening for anti-rVAR2 monoclonal antibody 5H4, where Mock is the blank control, wherein the detection system was the same as the experimental group except that no monoclonal antibody 5H4 was added; and HCS is hybridoma cell supernatant, that is the supernatant expressed by mouse B cell hybridoma;

(7) FIG. 7 (A) is a schematic diagram showing the binding mode among T cells expressing the chimeric antigen receptor of the present invention, VAR2CSA full-length protein (or its pl-CSA binding domain recombinant protein rVAR2), and tumor cells expressing pl-CSA epitope; and FIG. 7 (B) is a schematic diagram showing several different types of T cells constructed using the chimeric antigen receptor of the present invention and the binding mode among them and VAR2CSA full-length protein (or its pl-CSA binding domain recombinant protein rVAR2) and tumor cells expressing pl-CSA epitope;

(8) FIG. 8 is a plasmid map of the lentiviral expression vector of pLentiCART-anti-rVAR2 based on 5H4 single-chain antibody;

(9) FIG. 9 (A) is 10% SDS-PAGE protein gel electrophoresis detection of the stability of rVAR2 recombinant protein in human serum; and FIG. 9 (B) is Western blot detection of the stability of rVAR2 recombinant protein in human serum;

(10) FIG. 10 shows the lentiviral transfection efficiency of CART-anti-rVAR2 (5H4 ScFv) cells by flow cytometry and their expression in the extracellular antigen recognition domain (5H4 ScFv), where Mock is a T-cell group alone, where T cells are not treated with Alexa Fluor 647-labeled goat-anti-mouse F(ab)2 IgG antibody, Negative Control is a negative control, where T cells are treated with Alexa Fluor 647-labeled goat-anti-mouse F(ab)2 IgG antibody, and CART-anti-rVAR2 (5H4 ScFv) is a CAR-T cell group that expresses a chimeric antigen receptor of CART-anti-rVAR2 (5H4 ScFv) and that is obtained after a lentiviral vector expressing the chimeric antigen receptor is transfected into T cells;

(11) FIG. 11 (A) shows the detection of secretion levels of IL-2 in the process of incubation of sCART-anti-rVAR2 (5H4 ScFv) system with Raji cells; and FIG. 11 (B) shows the detection of secretion levels of IFN- in the process of incubation of sCART-anti-rVAR2 (5H4 ScFv) system with Raji cells;

(12) FIG. 12 shows the detection of the in-vitro ability of sCART-anti-rVAR2 (5H4 ScFv) system to kill tumor cells Raji, where the signal in the Far Red.sup.+ gate indicates the proportion of Raji cells, and the signal in the GFP.sup.+ gate indicates the proportion of CART-anti-rVAR2 (5H4 ScFv) cells; and

(13) FIG. 13 shows the in-vitro real-time dynamic monitoring of killing effects of sCART-anti-rVAR2 (5H4 ScFv) system on Raji cells, where cells marked by white arrows are tumor cells Raji labeled by Far Red, and cells not marked are CART-anti-rVAR2 (5H4 ScFv) cells co-expressing the GFP reporter gene.

DETAILED DESCRIPTION

(14) To further elaborate on the technical means adopted and the effects achieved in the present invention, the technical solutions of the present invention are further described below with reference to the drawings and specific embodiments, but the present invention is not limited to the scope of the embodiments.

Example 1: Expression and Purification of Recombinant Proteins Related to the pl-CSA Binding Domain of VAR2CSA Protein

(15) First, the DNA molecule encoding the amino acid sequence of the relevant recombinant protein (rVAR2) of the pl-CSA binding domain of the VAR2CSA protein was cloned into a prokaryotic or eukaryotic expression vector. Then the constructed prokaryotic expression vector was transduced into E. coli (such as BL21(DE3)), and the constructed eukaryotic expression vector was transduced into eukaryotic cells (such as human embryonic kidney cell line HEK293T containing large T antigen) respectively for protein expression. The protein expressing cells or medium supernatant was collected. Then corresponding protein tag affinity media was used for protein purification with steps as follows:

(16) Strep-Tag Affinity Purification System:

(17) First, E. coli expressing rVAR2 recombinant protein with Strep-tag was collected by centrifugation. Pre-cooled Buffer W (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) was added at 10 ml per gram of cells to re-suspend E. coli cells. The suspension was crushed 2-3 times with AH-1500 ultra-high pressure homogenizer (ATS Engineering Inc.) under a pressure of 100 Mpa at a low temperature to obtain cell lysate. The cell lysate supernatant was collected by centrifugation for 10 min at 14,000 rpm, 4 C. Strep-Tactin resin was washed with 2 resin volumes of Buffer W (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) and equilibrated. The collected cell lysate supernatant was incubated with the equilibrated Strep-Tactin resin for 30 min-1 h, and then allowed to pass through the column. After the penetrating solution was all flowed out, at least 5 Strep-Tactin resin volumes of pre-cooled Buffer W (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) was added portion-wise to wash the resin. 3 Strep-Tactin resin volume of pre-cooled Buffer E (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin) was added to elute and collect the rVAR2 recombinant protein with Strep-tag from the Strep-Tactin resin in 3 times. Then a certain amount of the purified sample of strep-tagged rVAR2 recombinant proteinwas analyzed by SDS-PAGE protein gel electrophoresis. The results were shown in FIG. 1(A). The predicted average molecular weight of the strep-tagged rVAR2 recombinant protein is about 74 kDa.

(18) The amino acid sequence of the rVAR2 recombinant protein with a Strep-tag is as follows (SEQ ID NO.38):

(19) TABLE-US-00020 MVHSNYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESG IASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCV IEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNL EKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGL PPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPS HEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFG KLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNST TCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVI ENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRW DQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHL IDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQ QCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGS ARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDLEVDLQGDHGLSAW SHPQFEK.

(20) The black and bold amino acid sequences are the amino acid sequences expressed by the vector backbone DNA introduced during the gene cloning process, and the underlined amino acid sequence is the Strep-tag sequence (SEQ ID NO.28). The rVAR2 recombinant protein with a Strep-tag was purified via the affinity of the Strep-tag II peptide and Strep-Tactin resin.

(21) 3FLAG-Tag Affinity Purification System:

(22) A eukaryotic (mammalian cell) protein expression vector with secreted signal peptide or non-secreted signal peptide was used to construct an expression vector of rVAR2 recombinant protein with 3FLAG-tag. The expression vector was transfected into HEK293 cells or HEK293T cells by means of electroporation or PEI transfection to express rVAR2 recombinant protein with 3FLAG-tag. Then the cell culture supernatant (for secreted expression vectors) or cells (for non-secretory vectors) were collected, respectively. 5% of pH-adjusted buffer (1 M Tris-HCl, 3 M NaCl, pH 7.4) was added to the cell culture supernatant for later use. A cell lysis bufferBuffer L (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, 1% TRITON X-100, and 1% protease Inhibitor (Sigma, Cat. #P8340)) was added to the collected cells at a ratio of 10.sup.6-10.sup.7 cells/ml, followed by incubation at room temperature for 30 min. Centrifuged at 12,000g, 4 C. for 10 min to collect the supernatant from cell lysate for later use.

(23) The prepared expression supernatant or cell lysate supernatant containing rVAR2 recombinant protein with a 3FLAG-tag was incubated with equilibrated affinity resin coupled to ANTI-FLAG M2 antibody or magnetic beads (Sigma) coupled to ANTI-FLAG M2 antibody on ice for 2 hours, and then allowed to pass through a column, or resin or magnetic beads binding to rVAR2 recombinant protein with a 3FLAG-tag were collected with a magnetic stand. The collected resin or magnetic beads were washed with 20 volumes of TBS buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.4) in three times, and eluted with 5 volumes of 3FLAG peptide eluent (prepared with TBS buffer, the 3FLAG peptide concentration was 150 ng/l), and rVAR2 recombinant protein with 3FLAG-tag was collected. Then a certain amount of the purified sample of rVAR2 recombinant protein with a 3FLAG-tag was analyzed by SDS-PAGE protein gel electrophoresis. The results were shown in FIG. 1(B). The predicted average molecular weight of the rVAR2 recombinant protein with a 3FLAG-tag is about 76 kDa, while 10% of the molecular weight detected by SDS-PAGE gel electrophoresis was about 115 kDa, indicating that there may be post-translational modification for the protein.

(24) The amino acid sequence of the rVAR2 recombinant protein with the 3FLAG-tag is as follows (SEQ ID NO.39):

(25) TABLE-US-00021 MVHSNYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESG IASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCV IEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNL EKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGL PPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPS HEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFG KLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNST TCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVI ENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRW DQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHL IDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQ QCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGS ARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDLEGGGGSGGGGSGG GGSADYKDHDGDYKDHDIDYKDDDDK.

(26) The black and bold amino acid sequences are the amino acid sequences expressed by the vector backbone DNA introduced during the gene cloning process, and the underlined amino acid sequence is the 3FLAG-tag sequence (SEQ ID NO.27). The rVAR2 recombinant protein with a 3FLAG-tag was purified via the affinity of the 3FLAG-tag and the resins or magnetic beads bound with the ANTI-FLAG M2 antibody.

Example 2: Obtaining Murine-Derived Polyclonal Antibodies and Monoclonal Antibodies Against rVAR2 Protein

(27) Three BALB/c mice aged 4-6 weeks were immunized with the prokaryotically expressed and purified rVAR2 recombinant protein. The specific steps are as follows: First, the purified rVAR2 recombinant protein was dialyzed 3 times against phosphate buffered saline (PBS, pH7.4) and the protein was concentrated to 1 g/l through ultrafiltration. 100 l of a solution of the rVAR2 recombinant protein (100 g) and 100 l of Freund's complete adjuvant were emulsified, and then mice were injected with the resultant at multiple points on the back for primary immunization. Then 100 l of a solution of the rVAR2 recombinant protein (100 g) and 100 l of Freund's complete adjuvant were emulsified, and then the mice were injected with the resultant at multiple points on the back for 2-3 booster immunization. Then enzyme-linked immunosorbent assay (ELISA) was used to detect the function and titer of the polyclonal antibody in the serum of immunized mice. The results are shown in FIG. 2. With an OD450 value of about 1.0 as the standard, the detected mouse antiserum (polyclonal) titer is about 1:25000.

(28) Appropriate amounts of feeder cells from mouse abdominal cavity and mouse myeloma cells SP2/0 were prepared for later use. Immunized BALB/c mice were anesthetized with isoflurane. Eyeballs were removed to collect blood. Serum was separated for use as a positive control serum during antibody detection. The immunized BALB/c mice were killed by breaking their necks. The spleen was surgically removed and placed in a petri dish containing 10 ml of incomplete culture medium. The spleen was washed gently and surrounding connective tissue was carefully peeled off. The isolated mouse spleen was transferred to another stainless steel mesh in a plate containing 10 ml of incomplete medium, and then ground into a cell suspension with a syringe needle core to allow the spleen cells to enter the incomplete medium in the plate. Pipetted several times with a pipette to yield a single cell suspension which was then counted. Usually 110.sup.8-2.510.sup.8 spleen cells were obtained for each mouse. 110.sup.8 spleen cells and 110.sup.7 myeloma cells SP2/0 were fused by PEG treatment to prepare hybridomas, and plated into 96-well plates containing feeder cells. Hybridoma cells were cultivated and screened using HAT medium (a cell culture medium containing three substances: hypoxantin, aminopterin and thymidin). Hybridoma cells were observed under microscope for growth, and the cell culture supernatant was drawn for antibody detection when they grew to more than 1/10 of the well bottom area. The hybridoma cells in the positive antibody detection well were monoclonalized by a limiting dilution method, and the cell culture supernatant was drawn for antibody detection again when they grew to more than 1/10 of the well bottom area. A monoclonal hybridoma cell strain secreting an antibody having a high affinity to the rVAR2 recombinant protein was selected. Part of the cells was cryopreserved. Some of the remaining cells were further cultured to collect supernatant containing anti-rVAR2 monoclonal antibody. Some of the cells were used to extract RNA, and the variable region coding sequences of the heavy and light chains of the murine IgG antibody were subsequently analyzed with RT-PCR using specific primers. Among them, a cell strain secreting monoclonal antibody 5H4 was selected. The coding sequences of the variable regions of its heavy chain and light chain are shown in Table 1, the amino acid sequences of the variable regions of the heavy chain and light chain of 5H4 are shown in Table 2, and the complementary determining region (CDR) sequences of the heavy chain and light chain of 5H4 are shown in Table 3.

(29) TABLE-US-00022 TABLE1 CodingDNAsequenceofheavychainvariable region(V.sub.H)andlightchainvariable region(V.sub.L)ofmonoclonalantibody5H4 Name DNAsequence V.sub.Hchain GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAG of5H4 CCTGGAGGGTCCCTAAAACTCTCCTGTGCAGCCTCTGGA (SEQID TTCACTTTCAGTAACTATGCCATGTCTTGGGTTCGCCAG NO.21) TCTCCAGAGAGGAGGCTGGAGTGGGTCGCAGAAATTAGT ATTACTGGTCGTTACACCTACTATCCAGACACTGTGACG GGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACC CTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACG GCCATGTATTATTGTACAAGGGAGGGATATGACTACGCC CCCTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTC ACTGTCTCTGCA V.sub.Lchain GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTC of5H4 AGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTGGT (SEQID CAGACCCTTGTACATCGTAATGGAATCACCTATTTAGAA NO.22) TGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTG ATCTACAAAGTTTCCAACCGATTTTCTGGAGTCCCAGAC AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTC AAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTAT TACTGCTTTCAAGGTTCACATGTTCCTCGGACGTTCGGT GGAGGCACCAAGCTGGAAATCAAA

(30) TABLE-US-00023 TABLE2 Aminoacidsequenceofheavychainvariable region(V.sub.H)andlightchainvariable region(V.sub.L)ofmonoclonalantibody5H4 Name Aminoacidsequence V.sub.Hchain EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQ of5H4 SPERRLEWVAEISITGRYTYYPDTVTGRFTISRDNAKNT (SEQID LYLEMSSLRSEDTAMYYCTREGYDYAPSWFAYWGQGTLV NO.19) TVSA V.sub.Lchain DVVMTQTPLSLPVSLGDQASISCRSGQTLVHRNGITYLE of5H4 WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTL (SEQID KISRVEAEDLGIYYCFQGSHVPRTFGGGTKLEIK NO.20)

(31) TABLE-US-00024 TABLE3 Complementarydeterminingregion(CDR)sequence ofheavychainvariableregion(V.sub.H)andlight chainvariableregion(V.sub.L)of monoclonalantibody5H4 Name CDR1 CDR2 CDR3 V.sub.Hchain GFTFSNYA ISITGRYT TREGYDYAPSWFAY of5H4 (SEQID (SEQID (SEQIDNO.15) NO.13) NO.14) V.sub.Lchain QTLVHRNGITY KVS(SEQID FQGSHVPRT(SEQ of5H4 (SEQID NO.17) IDNO.18) NO.16)

Example 3: Detection of Targetability of rVAR2 Protein to Tumor Cells Using Flow Cytometry

(32) 510.sup.5 cells of different types of in vitro isolated or cultured (including normal cell control group and tumor cell group) were incubated with purified prokaryotically or eukaryotically expressed rVAR2 protein, and then murine polyclonal antibody against rVAR2 protein obtained by immunizing mice, and the purchased FITC dye-labeled goat anti-mouse secondary antibody (IgG H&L, Abcam, cat #ab6785) for 45 minutes, respectively. The cells were washed 3 times with a 4 C. pre-cooled PBS solution containing 0.02% NaN.sub.3 and 2% FBS before incubated with the next reagent or after the reagent incubation was completed, with 2 min intervals. Finally, flow cytometry was used to detect the targetability of rVAR2 protein to several different types of tumor cells. The experimental groups are shown in Table 4 below:

(33) TABLE-US-00025 TABLE 4 Experimental groups in detection of targetability of rVAR2 protein to tumor cells using flow cytometry FITC-labeled Mouse goat anti- anti-mouse rVAR2 rVAR2 secondary protein polyclonal antibody (0.5 M) antibody (IgG H&L) CTR1 CTR2 + + EXP1 + + +

(34) The results are shown in FIG. 3. As can be seen from FIG. 3, rVAR2 protein can specifically target several different types of tumor cells. For example, human lung cancer cell lines, including NCI-H460 (large cell lung cancer cell line, ATCC #HTB177), NCI-H520 (squamous cell lung cancer cell line, ATCC #HTB182) and A549 (lung adenocarcinoma cell line, ATCC #CCL185) were specifically bound by rVAR2 protein to varying degrees. rVAR2 protein also could target and bind to human placental choriocarcinoma cell line BeWo (ATCC #CCL98). In addition, B-cell lymphoma cell line Raji (ATCC #CCL86) and acute bone marrow Leukemia cell line KG-1a (ATCC #CCL246.1) were also specifically bound by rVAR2 protein. However, the binding between rVAR2 protein and PBMC from healthy people, as well as the binding of rVAR2 protein to human umbilical vein endothelial cells (HUVEC, ATCC #PCS-100-010) were negative.

Example 4: Functional Verification of Murine Monoclonal Antibody 5H4 Against rVAR2 Recombinant Protein

(35) Western blot was used to verify the binding ability of monoclonal antibody 5H4 to its specific antigen of rVAR2 recombinant protein. The primary antibody was the purified monoclonal antibody 5H4, and the secondary antibody was HRP-labeled goat anti-mouse antibody. The results are shown in FIG. 4.

(36) It can be seen from FIG. 4 that the prokaryotically expressed and purified rVAR2 protein was about 74 kDa, and the eukaryotically expressed and purified rVAR2 protein was about 115 kDa. Western blot identification results showed that the monoclonal antibody 5H4 can bind to prokaryotically or eukaryotically expressed and purified rVAR2 protein.

Example 5: Screening of Epitope for Anti-rVAR2 Monoclonal Antibody 5H4

(37) According to the structural and functional properties of rVAR2 which is the core domain responsible for binding between VAR2CSA and placental-like chondroitin sulfate A(pl-CSA), it can be subdivided into three main domain components, including ID1, DBL2X and ID2a (Clausen et al., 2012). The amino acid sequence of rVAR2 protein and its domain components are as follows:

(38) The amino acid sequence of rVAR2 protein is as follows (SEQ ID NO.39):

(39) TABLE-US-00026 NYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESGIASV EQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHT SLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVL ASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRT QSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKK NDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFR KYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCG DGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCK SCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIY KRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIG LTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQQCNT AVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTM KRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD.

(40) The amino acid sequence of ID1 polypeptide is as follows (SEQ ID NO. 2):

(41) TABLE-US-00027 NYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESGIASV EQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHT SLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVL AS.

(42) The amino acid sequence of DBL2X polypeptide is as follows (SEQ ID NO.3):

(43) TABLE-US-00028 LTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQS LCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKND DNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKY IKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDG SVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSC KESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKR YSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIG.

(44) The amino acid sequence of ID2a polypeptide is as follows (SEQ ID NO. 4):

(45) TABLE-US-00029 LTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQQCNT AVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTM KRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD.

(46) The coding DNA sequences of ID1, DBL2X, and ID2a polypeptides were cloned into E. coli expression vectors. Proteins of the three were obtained after protein expression and purification. SDS-PAGE protein gel electrophoresis results are shown in FIG. 5. The purified ID2a protein with Strep-tag had a predicted average molecular weight of about 15 kDa; the purified DBL2X protein with Strep-tag had a predicted average molecular weight of 40 kDa; and the purified ID1 protein with Strep-tag a predicted average molecular weight of 17 kDa.

(47) The epitope for anti-rVAR2 monoclonal antibody 5H4 was preliminarily identified using an antigen-specific ELISA: The purified 3 antigen peptides including ID1, DBL2X, and ID2a were diluted with Na.sub.2CO.sub.3NaHCO.sub.3 buffer (pH 9.6) to a final concentration of 1 g/ml, respectively, added to 96-well microtiter plates at 100 l antigen/well, and incubated at 37 C. for 2 h. Washed twice with PBS-T buffer (PBS buffer containing 0.05% (v/v) Tween 20, pH7. 4). The liquid in the wells of the microtiter plate was slapped to dry. 250 l 5% (v/v) skim milk/PBS-T buffer was added to each well, and incubated at 37 C. for 1.5 h. Washed 4 times with PBS-T, and the liquid in the wells of the microtiter plate was slapped to dry. 100 l of monoclonal antibody 5H4 (primary antibody) purified from the supernatant of the hybridoma diluted in 0.1% (v/v) skim milk/PBS-T buffer was added to each well, and incubated at 37 C. for 60 min. After incubating with the primary antibody, the plate was washed 4 times with PBS-T, and the liquid in the wells of the microtiter plate was slapped to dry. 100 l of HRP-labeled goat anti-mouse IgG (secondary antibody) diluted with 0.1% (v/v) skim milk/PBS-T buffer at 1:8000 was added to each well, and incubated at 37 C. for 45 min. After incubating with the secondary antibody, the plate was washed 4 times with PBS-T, and the liquid in the wells of the microtiter plate was slapped to dry. 100 l TMB chromogenic substrate was added to each well, the plate was then incubated at 37 C. under darkness for 10 min, and then 50 l of 1 M sulfuric acid was added to each well to stop the reaction, and the absorbance of each well at 450 nm was measured.

(48) The ELISA results are shown in FIG. 6. Only the detection results of ID2a antigen peptide-coated wells are consistent with the detection results of rVAR2 protein-coated wells, indicating that the epitope of anti-rVAR2 monoclonal antibody 5H4 is located on the ID2a polypeptide epitope.

Example 6: Construction of CAR-T Cell and Functional Mechanism Thereof

(49) In order to further improve the killing activity of this broad-spectrum CAR-T cell based on VAR2CSA and enhance its application safety, a switchable CAR-T cell comprising VAR2CSA or its pl-CSA binding domain recombinant protein (rVAR2) and CAR-T (CART-anti-VAR2CSA or CART-anti-rVAR2) cell made of its single-chain antibody (anti-VAR2CSA ScFv or anti-rVAR2 ScFv) as a system was constructed: The full-length VAR2CSA protein or the rVAR2 protein was used to bind and mark tumor cells based on its targetability to tumor cells. Then, CART-anti-VAR2CSA or CART-anti-rVAR2 was constructed using murine single-chain antibody or humanized single-chain antibody that we developed and was specific for the full-length VAR2CSA protein or the rVAR2 protein. The mechanism of this system is to indirectly target and kill tumor cells marked by the full-length VAR2CSA protein or rVAR2 protein using CART-anti-VAR2CSA or CART-anti-rVAR2. A schematic diagram is shown in FIG. 7.

(50) The switchable CAR-T cell technology system constructed by the VAR2CSA full-length protein or the recombinant protein of its pl-CSA binding domain together with CAR-T cells constructed with its single-chain antibody is named sCART-anti-VAR2CSA (a system containing the full-length protein of VAR2CSA) or sCART-anti-rVAR2 cell (a system containing recombinant protein of the pl-CSA binding domain of VAR2CSA protein).

(51) Taking CART-anti-rVAR2 (5H4 ScFv) as an example, as shown in FIG. 8, its single-chain antibody ScFv sequence responsible for recognizing the ID2a epitope on rVAR2 includes the V.sub.H chain of anti-rVAR2 monoclonal antibody 5H4 and the V.sub.L chain of 5H4 as well as the connecting sequence L (Linker) between the two, wherein the linker sequence includes but is not limited to amino acid sequences such as GGGGSGGGGSGGGGS.

Example 7: Detection of Stability of rVAR2 Protein in Human Serum

(52) In order to confirm whether rVAR2 protein can be used as a navigation system of CART-anti-rVAR2 cells to target and kill tumor cells, it is first necessary to determine whether rVAR2 protein can stably exist in human blood.

(53) Therefore, about 1 ml of serum was separated from 2 ml of blood donated by a patient with non-small cell lung cancer (male, age: 64). Then to each well of a V-bottom 96-well plate, 25 l of patient's serum and 5 l rVAR2 recombinant protein which was filtered and sterilized by a 0.2 m protein low-adsorption filter membrane were added and mixed to a final concentration of the rVAR2 recombinant protein of 0.2 g/l. The plated was placed in a 37 C., 5% CO.sub.2 incubator for processing.

(54) Samples were collected according to the number of days the samples were placed in the incubator (Day). Day 0 means the 0th day, that is, 30 l of sample was collected immediately after mixing. Day 1 means that 30 l of sample that was processed for one day (24 h) in the 37 C., 5% CO.sub.2 incubator was collected, and so on. Samples on Day 2, Day 3, Day 4, Day 5, Day 7, Day 9, Day 11, Day 14, Day 17 and Day 22 were collected. Each sample was added with 570 l of SDS-PAGE protein loading buffer (2), boiled for 3 min, cooled and stored at 20 C. for later use. For negative control group (NC), 10 l of the lung cancer patient's serum was diluted to a total volume of 20 times with SDS-PAGE protein loading buffer (2), boiled for 3 min, cooled and stored at 20 C. for later use. For positive control group (PC), the purified rVAR2 recombinant protein was diluted with SDS-PAGE protein loading buffer (2) to 0.1 g/l, boiled for 3 minutes, cooled and stored at 20 C. for later use.

(55) Samples were divided into two groups. 5 l of the prepared samples were taken, respectively, and loaded on a 10% SDS-PAGE gel for electrophoresis. One group was transferred to a PVDF membrane, and the stability of rVAR2 recombinant protein with Strep-tag was detected through Western blot using an anti-Strep-tag monoclonal antibody.

(56) The results are shown in FIG. 9 (A) and FIG. 9 (B). The rVAR2 recombinant protein with a Strep-tag has an average molecular weight of about 74 kDa, and human serum albumin has an average molecular weight of about 66.5 kDa, indicating that under treatment conditions of 37 C., 5% CO.sub.2 incubator, rVAR2 recombinant protein can stably exist in the serum of lung cancer patients for more than 3 weeks.

Example 8: Construction of CART-Anti-rVAR2 Cells

(57) First, the chimeric antigen receptor (CAR) expression vector of CART-anti-rVAR2 cells were constructed. The 5H4 ScFv of the anti-ID2a domain of the rVAR2 recombinant protein was cloned into a lentiviral expression vector, where the vector was sequentially cascaded, via the EF1 promoter downstream, with the CD8 signaling peptide, 5H4 ScFv, CD8a hinge region (CD8 Hinge), CD28 transmembrane sequence (TM), CD28 costimulatory factor, CD137 (4-1BB) costimulatory factor and CD3 domain sequence, as shown in FIG. 8.

(58) The large amount of extracted CART-anti-rVAR2 (5H4 ScFv) expression plasmids were mixed with the third-generation lentiviral packaging plasmids pMDLg-pRRE:pRSV-Rev:pMD2.G with a mass ratio 3:1:1, and co-transfected to HEK293T cells by a PEI transfection method. After 72 h of transfection, the culture supernatant was collected, and then the collected culture supernatant was filtered by a 0.45 m membrane filter, such filtered solutions were then concentrated by ultrafiltration to a lentivirus titer of 1.610.sup.8 TU/ml to 110.sup.10 TU/ml, and stored at 80 C. in the refrigerator for later use. The CART-anti-rVAR2 (5H4 ScFv) cells were produced according to the following steps.

(59) (1) Isolation of PBMC Cells

(60) 50 ml of whole blood donated by a volunteer was centrifuged at 800 g for 10 min. A white cell layer was taken out and then diluted to 8 ml with 2% FBS (Fetal Bovine Serum) containing PBS buffer. 4 ml of LymphoPrep was pipetted into a 15 ml centrifuge tube, and then the diluted blood was gently added along the tube wall onto the layered solution, keeping the interface between the two solution layers clear. The resulting mixture was centrifuged at 800 g for 20 min (acceleration was set to 6 and deceleration was set to 1). Off-white mononuclear cells were gently pipetted with a Pasteur pipette, added to another centrifuge tube that already contained 10 ml of RF-10 (RPMI 1640 medium with 10% inactivated FBS), and then mixed well. The above resulting mixture was centrifuged at 500 g for 5 min, and the supernatant was discarded. After that, the cells were resuspended by adding 10 ml of RF-10, 20 microliters of cell suspension was stained with Trypan blue and counted. Then the cells were centrifuged at 350 g for 10 min, and the supernatant was discarded.

(61) (2) Magnetic Activated Cell Sorting of CD4.sup.+ T and CD8.sup.+ T Cells

(62) CD4.sup.+ T cells and CD8.sup.+ T cells in PBMC were isolated respectively by using the Dynabeads CD4 Positive Isolation Kit (Invitrogen, Cat. #11331D) and the Dynabeads CD8 Positive Isolation Kit (Invitrogen, Cat. #11333D). The basic operation steps of the isolation were as follows. Magnetic beads (Dynabeads) were vortexed well for resuspension. 25 l of Dynabeads was transferred to a tube and mixed well with 1 ml of added Buffer 1. Place the tube in a magnet for 1 min and discard the supernatant. 25 l of Buffer 1 was added to suspend the magnetic beads for later use. PBMC cells were resuspended with Buffer 1 to a density of 110.sup.7 cells/ml. 25 l of washed Dynabeads CD4 was added to 1 ml of PBMC cells, incubated at 2 to 8 C. for 20 min, and placed on a shaker and rotated at an angle. The tube was placed on the magnet for 2 min, and the supernatant was collected. The tube was removed from the magnet, 1 ml of Buffer 1 was added into the tube and pipetted and mixed, the tube was placed back on the magnetic stand for 2 min, and the supernatant was collected; the above steps were repeated again, the supernatant containing other cells collected in the above steps was transferred to a new sterile tube for subsequent isolation of CD8.sup.+ T cells. 100 l Buffer 2 was added to resuspend the CD4.sup.+ T cells combined by the magnetic beads, 10 l of DETACHaBEAD was added, and the above mixture was incubated at room temperature for 45 min to release the cells from the Dynabeads. The tube was placed on the magnet for 1 min, and the supernatant containing the cells was transferred to a new tube. 500 l of Buffer 2 was added to the new tube to wash the magnetic beads 2 to 3 times, and the supernatant containing part of CD4.sup.+ T cells was collected. 4 ml of Buffer 2 was added and centrifuged at 350 g for 5 min, the supernatant containing DETACHaBEAD was removed, and the collected CD4.sup.+ T cells were resuspended with Buffer 2. CD8.sup.+ T cells were subsequently collected from the supernatant collected in the process of CD4.sup.+ T cell sorting according to the operation instructions on the kit.

(63) (3) Culture of T Cells

(64) CD4.sup.+ T and CD8.sup.+ T cells sorted by magnetic beads were centrifuged at 350 g for 10 min. The centrifuged cells were resuspended with RF-10 (RPMI 1640 medium containing 10% inactivated FBS) and counted. CD4.sup.+ T and CD8.sup.+ T cells were added at a ratio of 1:1 in a cell culture plate with a cell density of 510.sup.5 cells/ml and cultured. CD3/CD28 antibody magnetic beads were added to a T-cell special medium, where a ratio of the quantity of added magnetic beads to the cells was 1:1. Recombinant human interleukin-2 (rhIL-2) was added to make the final concentration 10 ng/ml. The cells were counted 2 to 3 times a week, and the cell proliferation was recorded.

(65) (4) Lentiviral Transfection of T Cells

(66) CD4.sup.+ T and CD8.sup.+ T cells sorted by magnetic beads were centrifuged at 350 g for 10 min. The centrifuged cells were resuspended with an added complete medium and then counted. CD4.sup.+ T and CD8.sup.+ T cells were added at a ratio of 1:1 in a 96-well plate and cultured with a cell density of 510.sup.5 cells/ml and 110.sup.5 cells per well. The magnetic beads were washed and added to a culture dish with a ratio of 1:1 to the cells. Recombinant human IL-2 (rhIL-2) was added to make the final concentration 10 g/l. The cells were stimulated for 24 h and then infected. Polybrene was added to make the final concentration 6 g/ml. The above mixture was mixed well. 16 to 24 h later, the medium was changed. After 3 days, the T cell infection efficiency was detected by flow cytometry. The results are shown in FIG. 10. It can be seen that the infection efficiency detected by flow cytometry was about 45%, which can make the preparation of CAR-T cells more efficiently.

Example 9: Cytokine Secretion Detection in the Process of the Co-Incubation of Switchable CAR-T Cells, sCART-Anti-rVAR2 System, with Tumor Cells

(67) In order to detect whether switchable CAR-T cells, sCART-anti-rVAR2 system, had a potential killing effect on tumor cells, first, the cytokine secretion level in the process of the co-incubation of the sCART-anti-rVAR2 system with tumor cells was detected by using the relevant cytokine detection kit [R&D Systems, Human IFN- ELISA detection kit (Cat. #dif50) and Human IL-2 ELISA detection kit (Cat. #d2050)] according to the following steps.

(68) (1) Collection and Pretreatment of Cells

(69) Effector cells [CAR-T cells: CART-anti-rVAR2 (5H4 ScFv)] and target cells (cancer cells) were collected separately and centrifuged at 250g for 4 min, and the supernatant was removed. The cells were washed with 10 ml of serum-free 1640 medium twice, and then the cell density was adjusted with the 1640 medium to 610.sup.5 cells/ml.

(70) (2) Experimental Grouping

(71) Experimental group: effector cells [CART-anti-rVAR2 (5H4 ScFv), CARTV2] and target cells (Raji, R) were evenly spread in a 48-well plate at a ratio of 1:1, where each type of cells was set to 1.2510.sup.5/well/250 that is, the total volume was 500 where in the pre-coincubation group of effector cells (or target cells) and rVAR2 recombinant proteins (rVAR2, V), rVAR2 proteins and effector cells (or target cells) were incubated at 37 C. for 1 h before subsequent operations. The above groups were respectively marked as: [CARTV2+V]+R, which means that effector cells were first co-incubated with rVAR2 recombinant proteins, and then the target cells were added; or [V+R]+CARTV2, which means that rVAR2 recombinant proteins were first co-incubated with the target cells, and then effector cells were added. In this experiment, the concentration of rVAR2 recombinant proteins used herein was 18 nM.

(72) Control group: T+R group, namely the group of normal T Cells (T) plus target cells, where each cell was set to 1.2510.sup.5 cells/well/250 the insufficient part was made up with 250 l of RPMI-1640 culture medium, and the total volume was kept at 500 l; CARTV2+R group, namely the group of effector cells plus target cells; [T+V]+R group, that is, normal T cells were co-incubated with rVAR2 recombinant proteins, and then added to a target cell group; and [V+R]+T group, that is, rVAR2 recombinant proteins were co-incubated with target cells, and then added to a normal T cell group; where after cells in each group were incubated at 37 C. in an incubator for 24 h, the supernatant was collected for detection.
(3) Detection of Cytokines 1) 100 l of Assay Diluent was added to each well of the ELISA plate. 2) Samples and the concentration-gradient-diluted standards were added to the ELISA well plate that had been coated with antibody, with 100 l per well, and incubated at room temperature for 2 h after air bubbles were removed. 3) The liquid in the well was removed, 300 l of rinsing buffer was added to each well to wash the mixture in the well three times, and the rinsing buffer was bolted up at the last time of washing. 4) 200 l of corresponding detection antibody was added and incubated for 2 h at room temperature. 5) Step 3) was repeated. 6) 200 l of substrate solution (A+B) was added and incubated for 30 min at room temperature in the dark. 7) 50 l of stop solution was added, and the solution would change from blue to yellow (if the color was green or the color was not changed, tap it to mix well). 8) The absorbance was detected at 450 nm by using a microplate reader. 9) The sample concentration was calculated based on the standard curve.

(73) The detection results are shown in FIG. 11 (A) and FIG. 11 (B). As shown in the above figures, a large amount of IFN- and IL-2 were secreted in the process of the co-incubation of sCART-anti-rVAR2 system with the tumor cell of B-cell lymphoma cell line Raji (ATCC #CCL86) or the co-incubation of sCART-anti-rVAR2 system with the large cell lung cancer cell line (NCI-H460, ATCC #HTB177), which improves anti-tumor activity, and also indicates that the sCART-anti-rVAR2 system has a potential killing effect on different types of tumor cells.

Example 10: Validation of Killing Effects of Switchable CAR-T Cells, sCART-Anti-rVAR2 System, on Tumor Cells by a Far Red Method

(74) First, the target cells were labeled by the Far Red method, namely, the CellTrace Far Red Cell Proliferation Kit (Invitrogen, Cat. #C34564) was used for labeling of the target cells. (1) An appropriate amount of tumor cells were taken and centrifuged at 300g for 3 min, and the supernatant was removed. (2) After the cells were resuspended with PBS buffer, the cells were centrifuged at 300g for 3 min, the supernatant was removed, and this step was repeated once. (3) The cells were resuspended with PBS buffer to a cell density of 110.sup.6 cells/ml. (4) 1 l of Far-Red at a concentration of 200 M was added to each 1 ml of cells, and incubated in a water bath at 37 C. for 20 min. (6) After the incubation, 5 times volume of RF-10 medium (RPMI 1640 medium containing 10% inactivated FBS) was added to stop the reaction in 5 min. (7) The cells were centrifuged at 300 g for 3 min, and the supernatant was removed. (8) The cells were resuspended to 510.sup.5/ml with the T cell expansion medium.

(75) Then, CAR-T cells were taken and centrifuged at 300g for 3 min, and then the supernatant was removed; after the cells were resuspended with PBS buffer, the cells were centrifuged at 300g for 3 min, and the supernatant was removed, and the above steps were repeated once; and the cells were resuspended to a density of 510.sup.5 cells/ml with the T cell expansion medium (Gibco, Cat. #A10485).

(76) In order to validate the ability of effector cells [CAR-T cells: CART-anti-rVAR2 (5H4 ScFv)] to kill target cells (cancer cells Raji) under the navigation of rVAR2 recombinant protein, the experiment was divided into the following groups, specifically as shown in Table 5. In each group, no matter which cell the rVAR2 recombinant proteins were co-incubated with first, the molar concentration of the proteins was 500 times that of the co-incubated corresponding cells, and the ratio of effector cells and target cells was 2:1.

(77) TABLE-US-00030 TABLE 5 Experimental grouping for detection of killing effects of sCART-anti-rVAR2 (5H4 ScFv) system on tumor cells by the Far Red method Group Full name Note V + R rVAR2 + Raji rVAR2 recombinant proteins were incubated with Raji cells in an incubator at 37 C. with 5% CO.sub.2. [T + V] + R [T cells + rVAR2] + Raji Normal T cells were co-incubated with rVAR2 recombinant proteins in an incubator at 37 C. with 5% CO.sub.2 for 30 min, and then added to Raji cells to co-incubate. [V + R] + T [rVAR2 + Raji] + T cells rVAR2 recombinant proteins were co-incubated with Raji cells in an incubator at 37 C. with 5% CO.sub.2 for 30 min, and then added to normal T cells to co-incubate. CART19 + R CART-CD19+ + Raji CAR-T cells constructed based on the single chain fragment variable antibody (ScFv) of the CD19 antigen were co-incubated with Raji cells in an incubator at 37 C. with 5% CO.sub.2, as the positive control. [CARTV2 + [CART-anti-rVAR2 CART-anti-rVAR2 (5H4 ScFv) cells were V] + R (5H4 ScFv) + rVAR2] + co-incubated with rVAR2 recombinant proteins in Raji an incubator at 37 C. with 5% CO.sub.2 for 30 min, and then added to Raji cells to co-incubate. [V + R] + [rVAR2 + Raji] + rVAR2 recombinant proteins were co-incubated CARTV2 CART-anti-rVAR2 with Raji cells in an incubator at 37 C. with 5% (5H4 ScFv) CO.sub.2 for 30 min, and then added to CART-anti-rVAR2 (5H4 ScFv) cells to co-incubate.

(78) After 16 h of incubation in the incubator at 37 C. with 5% CO.sub.2, the cells were centrifuged at 300 g for 3 min and then collected; the centrifuged cells were washed twice with PBS buffer containing 2% inactivated FBS and 0.02% NaN.sub.3, and then the cells were resuspended with the PBS buffer; and the Far-Red fluorescence signals were detected by flow cytometry (excitation wavelength of 630 nm and emission wavelength of 661 nm).

(79) The results of in vitro experiments are shown in FIG. 12, and the results indicate that the sCART-anti-rVAR2 (5H4 ScFv) system had killing effects on Raji cells, and as long as in the presence of rVAR2 recombinant proteins and T cells, it had a certain killing effect on Raji cells, from which it is speculated that normal T cells can also recognize and kill target cells labeled with rVAR2 recombinant protein.

Example 11: In-Vitro Real-Time Dynamic Monitoring of Killing Effects of Switchable CAR-T Cells, sCART-Anti-rVAR2 System, on Tumor Cells

(80) First, the target cells Raji were stained and labeled by using the same Far-Red method as in Example 10, and then the Raji cells were resuspended with the corresponding T cell culture medium and plated into a 96-well culture plate at a density of 5000 cells/100 l per well. The prepared CART-anti-rVAR2 (5H4 ScFv) cells were added at the effector-target ratio of 4:1. Since the lentiviral transfection efficiency was about 45% or more, the effector-target ratio as from 2:1 to 4:1. Two groups were divided according to the order of adding the rVAR2 recombinant proteins as the navigator between the effector cells and target cells: the rVAR2 recombinant proteins were co-incubated with CART-anti-rVAR2 cells (the final concentration of rVAR2 recombinant proteins was 18 nM, and CART-anti-rVAR2 cells were at a density of 20,000 cells/100 l) at 37 C. for 1 h and then added to tumor cells, and this group was named [CART-anti-rVAR2 (5H4 ScFv)+rVAR2]+Raji group; or rVAR2 recombinant proteins (the final concentration of rVAR2 recombinant proteins was 18 nM as well) were co-incubated with Raji cells at 37 C. for 1 h, and then added to CART-anti-rVAR2 cells at a density of 20,000 cells/100 l, and this group was named [Raji+rVAR2]+CART-anti-rVAR2 (5H4 ScFv) group. In addition, since Raji is a B cell lymphoma cell line with high cell surface expression of CD19, CD19-targeting CAR-T cells (CART-CD19.sup.+ cells, Porter et al., N Engl J Med. 2011, 365 (8): 725-33; Grupp et al., N Engl J Med. 2013; 368 (16): 1509-18.) could be used as positive control to measure the effects of the sCART-anti-rVAR2 (5H4 ScFv) system, so CART-CD19.sup.++Raji with the same cell number and effector-target ratio was set as a positive control group.

(81) All experimental groups and control groups were placed in the incubator and cultured at 37 C. with 5% CO.sub.2, and the killing activity of sCART-anti-rVAR2 (5H4 ScFv) system on tumor cells was observed in real time by the JuLI Stage automated live cell imaging system.

(82) The results are shown in FIG. 13. The results indicate that the switch-mediated CAR-T cell system of sCART-anti-rVAR2 (5H4 ScFv) had significant in-vitro killing effects on the tumor cells Raji, but the killing effects required longer time than CART-CD19.sup.+, which indicates that its killing effect was milder, and it may help to reduce the toxic side effects of CAR-T cells and enhance the safety of CAR-T cells.

(83) The applicant has stated that although the detailed method of the present invention is described through the embodiments described above, the present invention is not limited to the detailed method described above, which means that implementation of the present invention does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present invention, equivalent replacements of various raw materials of the product, the addition of adjuvant ingredients, and the selection of specific manners, etc. in the present invention all fall within the protection scope and the scope of disclosure of the present invention.