Chimeric Antigen Receptor, Construction Method Therefor and Application Thereof

Abstract

Provided are a chimeric antigen receptor, a construction method therefor and an application thereof. The chimeric antigen receptor consists of an antigen binding domain, an extracellular hinge region, a transmembrane domain, a co-stimulatory domain and a CD3z signaling domain. Further provided is a CAR-T cell which comprises two chimeric antigen receptors containing different antigen binding domains, and which is bispecific and exhibits increased cell killing efficiency and a better tumor inhibitory effect in vivo.

Claims

1. A chimeric antigen receptor composed of an antigen binding domain, an extracellular hinge region, a transmembrane domain, a co-stimulatory domain and a CD3z signaling domain.

2. The chimeric antigen receptor according to claim 1, wherein the extracellular hinge region is any one selected from the group consisting of CD8 extracellular hinge region (CD8hinge), CD28 extracellular hinge region (CD28hinge), ICOS extracellular hinge region (ICOShinge) and IgG4mt10+N297A extracellular hinge region (IgG4mt10+N297Ahinge); the transmembrane domain is any one selected from the group consisting of CD8 transmembrane domain (CD8TM), CD28 transmembrane domain (CD28TM) and ICOS transmembrane domain (ICOSTM); and the co-stimulatory domain is any one selected from the group consisting of 4-1BB co-stimulatory domain (4-1BBCSD), CD28 co-stimulatory domain (CD28CSD), ICOS co-stimulatory domain (ICOSCSD) and OX40 co-stimulatory domain (OX40CSD).

3. The chimeric antigen receptor according to claim 2, wherein structures comprising the extracellular hinge region, the transmembrane domain and the co-stimulatory domain are respectively as follows: CD8hinge-CD8TM-4-1BBCSD, CD28hinge-CD28TM-CD28CSD, ICOShinge-ICOSTM-ICOSCSD, CD28hinge-CD28TM-OX40CSD, IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD, IgG4mt10+N297Ahinge-CD28 TM-CD28CSD or IgG4mt10.sup.+N297Ahinge-ICOSTM-ICOSCSD.

4. The chimeric antigen receptor according to claim 3, wherein an amino acid sequence of the IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD is SEQ ID NO: 36; an amino acid sequence of the IgG4mt10+N297Ahinge-CD28TM-CD28CSD is SEQ ID NO: 37; and an amino acid sequence of the IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD is SEQ ID NO: 38.

5. The chimeric antigen receptor according to claim 2, wherein the antigen binding domain is a single-chain antibody (scFv) or a single domain antibody (sdAb).

6. The chimeric antigen receptor according to claim 5, wherein the antigen binding domain recognizes CD20 or recognizes CD22.

7. The chimeric antigen receptor according to claim 6, wherein the antigen binding domain is Leu16, said Leu16 is a humanized scFv recognizing CD20 and an amino acid sequence of said Leu16 is as set forth in SEQ ID NO: 3.

8. The chimeric antigen receptor according to claim 6, wherein the antigen binding domain is M971, said M971 is an scFv recognizing CD22 and an amino acid sequence of said M971 is as set forth in SEQ ID NO: 7.

9. A chimeric antigen receptor T cell (CAR-T cell), wherein the CAR-T cell expresses the chimeric antigen receptor of claim 1.

10. The chimeric antigen receptor T cell (CAR-T cell) according to claim 9, wherein the CAR-T cell expresses two chimeric antigen receptors comprising different antigen binding domains.

11. The CAR-T cell according to claim 10, wherein the two independent chimeric antigen receptors are CAR19 and CAR20, respectively; wherein said CAR19 recognizes CD19, and said CAR20 recognizes CD20.

12. The CAR-T cell according to claim 10, wherein the two independent chimeric antigen receptors are CAR19 and CAR22, respectively; wherein said CAR19 recognizes CD19, and said CAR22 recognizes CD22.

13. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A host cell, the host cell comprising the vector of claim 14.

16. A pharmaceutical composition comprising a pharmaceutically acceptable vector and the chimeric antigen receptor of claim 1.

17. (canceled)

18. (canceled)

19. A method for preparing a CAR-T cell, wherein the CAR-T cell expresses the chimeric antigen receptor of claim 1, and the method comprises the following step: Introducing a nucleic acid molecule encoding the chimeric antigen receptor into a T cell so as to obtain the CAR-T cell.

20. A method of treating tumor in a subject, comprising administering to the subject a chimeric antigen receptor T cell (CAR-T cell) according to claim 9.

21. The method according to claim 20, wherein the tumor is a hematological tumor, preferably, the hematological tumor is B-cell malignancy, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma, mastocytoma or follicular lymphoma.

22. A method of treating tumor in a subject, comprising administering to the subject a nucleic acid molecule encoding a chimeric antigen receptor according to claim 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows the schematic diagram of the structures of a tandem bispecific CAR-T and a combinational bispecific CAR-T.

[0042] FIG. 2 shows the schematic diagram of the structures of 7 kinds of combinational bispecific chimeric antigen receptor CAR19-CAR20s.

[0043] FIG. 3 shows the killing efficiency of 7 kinds of CAR19-CAR20-T cells comprising bispecific chimeric antigen receptors with different structures for CD19.sup.+K562-luc-GFP target cells.

[0044] FIG. 4 shows the killing efficiency of 7 kinds of CAR19-CAR20-T cells comprising bispecific chimeric antigen receptors with different structures for CD20.sup.+K562-luc-GFP target cells.

[0045] FIG. 5 shows the T cell phenotypes of 7 kinds of CAR19-CAR20-T cells comprising bispecific chimeric antigen receptors with different structures.

[0046] FIG. 6 shows the results illustrating CAR19.sup.+CAR20.sup.+ dual-positive population expressed by CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153.

[0047] FIG. 7 shows the schematic diagram of the dosage regimen described in the validation test of the in-vivo tumor inhibitory activity of CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 in mice.

[0048] FIG. 8 shows the survival rates of the tumor-bearing mice in Group G1, Group G3 and Group G4 after being respectively administered with PBS, a vector that comprises the nucleic acid molecule encoding PCTL152 CAR19-CAR20, and a vector that comprises the nucleic acid molecule encoding PCTL153 CAR19-CAR20.

[0049] FIG. 9 shows the schematic diagram of the dosage regimen described in the validation test of the in-vivo tumor inhibitory activity of CAR19-CAR20-T cells comprising PCTL153, single-targeting CAR19-T cells, single-targeting CAR20-T cells and tandem CAR20-19-T cells in mice.

[0050] FIG. 10 shows the survival rates of the tumor-bearing mice in Group G1, Group G3, Group G4, Group G5 and Group G7 after being respectively administered with PBS, a vector that comprises the nucleic acid molecule encoding CAR-19, a vector that comprises the nucleic acid molecule encoding CAR-20, a vector that comprises the nucleic acid molecule encoding the combinational bispecific chimeric antigen receptor PCTL153 CAR19-CAR20, and a vector that comprises the nucleic acid molecule encoding the tandem bispecific chimeric antigen receptor CAR20-19.

[0051] FIG. 11 shows the killing efficiency of 7 kinds of CAR19-CAR22-T cells comprising bispecific chimeric antigen receptors with different structures for CD19.sup.+K562-luc-GFP target cells.

[0052] FIG. 12 shows the killing efficiency of 7 kinds of CAR19-CAR22-T cells comprising bispecific chimeric antigen receptors with different structures for CD22.sup.+K562-luc-GFP target cells.

DETAILED DESCRIPTION

[0053] The technical solutions of the present disclosure are further illustrated below by means of specific embodiments. It should be emphasized that the present disclosure is not limited to the specific embodiments exemplified and illustrated. In addition, titles of any section used herein are merely for purpose of organization, and are not to be construed as limiting the subject matters described.

[0054] Unless otherwise defined herein, scientific and technical terms used in the present disclosure will have the meanings commonly understood by one of ordinary skill in the art. In addition, unless otherwise required in the context, the terms in singular form should include the plural form thereof, and the terms in plural form should include the singular form thereof.

[0055] More specifically, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. In the present application, unless otherwise indicated, “or” is used to denote “and/or”. In addition, the use of the term “comprising” and other forms (such as “including” and “containing”) is not restrictive. In addition, the ranges provided in the specification and the appended claims include the endpoints and all values between the endpoints.

Example 1: Design of Combinational Bispecific Chimeric Antigen Receptors CAR19-CAR20 and CAR19-CAR22

[0056] The inventors designed 7 kinds of different CAR19-CAR20 combinational bispecific chimeric antigen receptors, wherein the structure of CAR19 was kept constant, that is, CAR19 with a structure of FMC63-CD8 hinge-CD8 TM-4-1BB-CD3z was selected (please refer to CN105392888A) and was combined with 7 kinds of CAR20s with different structures. Among them, the amino acid sequence of the antigen binding domain FMC63 in the above-mentioned CAR19 was as set forth in SEQ ID NO: 5, and the nucleotide sequence encoding this amino acid sequence was as set forth in SEQ ID NO: 6. The amino acid sequences of CD8 hinge, CD8 TM, 4-1BB and CD3z in the above-mentioned CAR19 were as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

[0057] The seven different CAR20s included Leu16-CD8 hinge-CD8 TM-4-1BBCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL126), Leu16-CD28 hinge-CD28 TM-CD28CSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL137), Leu16-ICOS hinge-ICOSTM-ICOSCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL138), Leu16-CD28 hinge-CD28 TM-OX40CSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL139), Leu16-IgG4mt10+N297A hinge-CD8 TM-4-1BBCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL151), Leu16-IgG4mt10+N297A hinge-CD28 TM-CD28CSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL152), Leu16-IgG4mt10+N297A hinge-ICOSTM-ICOSCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR20 and the above-mentioned CAR19 was referred to as PCTL153). The compositions of the seven different CAR20s were as shown in Table 1.

TABLE-US-00001 TABLE 1 Compositions of seven different CAR20s PCTL126 PCTL137 PCTL138 PCTL139 PCTL151 PCTL152 PCTL153 Antigen binding scFv Leu16 Leu16 Leu16 Leu16 Leu16 Leu16 Leu16 domain Hinge region Hinge CD8 CD28 ICOS CD28 IgG4mt10 + IgG4mt10 + IgG4mt10 + N297A N297A N297A Transmembrane Transmembrane CD8 CD28 ICOS CD28 CD8 CD28 ICOS domain Co-stimulatory costimulatory 4-1BB CD28 ICOS OX40 4-1BB CD28 ICOS domain domain Intracellular signaling domain CD3z CD3z CD3z CD3z CD3z CD3z CD3z signaling domain

[0058] To be specific, all the scFvs in the seven different CAR20s in Table 1 were a murine scFv (wherein the amino acid sequence of the murine scFv was as set forth in SEQ ID NO: 1, and the nucleotide sequence encoding this amino acid sequence was as set forth in SEQ ID NO: 2) humanized by conventional molecular biological means, and the humanized scFv was named Leu16, the amino acid sequence of which was as set forth in SEQ ID NO:3, and the nucleotide sequence encoding this amino acid sequence was as set forth in SEQ ID NO: 4.

[0059] There were four different options for the hinge region of the CAR20, i.e., CD8hinge, CD28hinge, ICOShinge or IgG4mt10+N297Ahinge, their amino acid sequences were as set forth in SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 31, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 24 and SEQ ID NO: 32, respectively. Among them, the hinge region as shown by IgG4mt10+N297Ahinge was a hinge region that was derived from natural IgG4 and had 8 mutated amino acids. To be specific, in natural IgG4, the amino acid S at position 228 was substituted with P, the amino acid E at position 233 was substituted with P, the amino acid F at position 234 was substituted with V, the amino acid L at position 235 was substituted with A, the amino acid D at position 265 was substituted with A, the amino acid N at position 297 was substituted with A, the amino acid L at position 309 was substituted with V, and the amino acid R at position 409 was substituted with K, so as to deprive the binding ability of FcγR (Fc gamma receptor), avoid the antibody-dependent cell-mediated cytotoxicity (ADCC) and the complement-dependent cytotoxicity (CDC), thereby effectively enhancing the in-vivo activity of CAR-T cells.

[0060] There were three different options for the transmembrane domain of the CAR20, i.e., CD8TM, CD28TM or ICOSTM, their amino acid sequences were as set forth in SEQ ID NO: 11, SEQ ID NO: 19 and SEQ ID NO: 25, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 12, SEQ ID NO: 20 SEQ ID NO: 26, respectively.

[0061] There were four different options for the co-stimulator domain of the CAR20, i.e., 4-1BBCSD, CD28CSD, ICOSCSD and OX40CSD, their amino acid sequences were as set forth in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 29, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 30, respectively. That is, the CAR20s in seven bispecific chimeric antigen receptor CAR19-CAR20s encoded the same antigen binding domain (i.e., having the same scFv) and CD3z signaling domain, wherein the amino acid sequence of the CD3z signaling domain was as set forth in SEQ ID NO: 15 (the nucleotide sequence encoding this amino acid sequence was as set forth in SEQ ID NO: 16). The only difference between the seven constructs was the different combinations of the hinge region, the transmembrane domain and the co-stimulator domain in CAR20. The schematic diagram of the structures of 7 kinds of CAR19-CAR20 combinational bispecific chimeric antigen receptors was as shown in FIG. 2.

[0062] The inventors also designed 7 kinds of different CAR19-CAR22 combinational bispecific chimeric antigen receptors, wherein the structure of CAR19 was kept constant, that is, CAR19 with a structure of FMC63-CD8 hinge-CD8 TM-4-1BB-CD3z was selected and was combined with 7 kinds of CAR22s with different structures. The seven different CAR22 included M971-CD8 hinge-CD8 TM-4-1BBCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL81), M971-CD28 hinge-CD28 TM-CD28CSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL103), M971-ICOS hinge-ICOSTM-ICOSCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL105), M971-CD28 hinge-CD28 TM-OX40CSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL124), M971-IgG4mt10+N297A hinge-CD8 TM-4-1BBCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL148), M971-IgG4mt10+N297A hinge-CD28 TM-CD28CSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL149), and M971-IgG4mt10+N297A hinge-ICOSTM-ICOSCSD-CD3z (the bispecific chimeric antigen receptor comprising both this CAR22 and the above-mentioned CAR19 was referred to as PCTL150). The compositions of the seven different CAR22s were as shown in Table 2.

TABLE-US-00002 TABLE 2 Compositions of seven different CAR22s PCTL181 PCTL103 PCTL105 PCTL124 PCTL148 PCTL149 PCTL150 Antigen binding scFv M971 M971 M971 M971 M971 M971 M971 domain Hinge region Hinge CD8 CD28 ICOS CD28 IgG4mt10 + IgG4mt10 + IgG4mt10 + N297A N297A N297A Transmembrane Transmembrane CD8 CD28 ICOS CD28 CD8 CD28 ICOS domain Co-stimulatory costimulatory 4-1BB CD28 ICOS OX40 4-1BB CD28 ICOS domain domain Intracellular signaling domain CD3z CD3z CD3z CD3z CD3z CD3z CD3z signaling domain

[0063] To be specific, the seven different CAR22s in Table 2 had an scFv with an amino acid sequence as set forth in SEQ ID NO: 7, and the nucleotide sequence encoding this amino acid sequence was as set forth in SEQ ID NO: 8. There were four different options for the hinge region of the CAR22, i.e., CD8hinge, CD28hinge, ICOShinge or IgG4mt10+N297Ahinge, their amino acid sequences were as set forth in SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 31, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 24 and SEQ ID NO: 32, respectively. Among them, the hinge region as shown by IgG4mt10+N297A was a hinge region that was derived from natural IgG4 and has 8 mutated amino acids. To be specific, in natural IgG4, the amino acid S at position 228 was substituted with P, the amino acid E at position 233 was substituted with P, the amino acid F at position 234 was substituted with V, the amino acid L at position 235 was substituted with A, the amino acid D at position 265 was substituted with A, the amino acid N at position 297 was substituted with A, the amino acid L at position 309 was substituted with V, the amino acid R at position 409 was substituted with K, so as to deprive the binding ability of FcγR (Fc gamma receptor), avoid the antibody-dependent cell-mediated cytotoxicity (ADCC) and the complement-dependent cytotoxicity (CDC), thereby effectively enhancing the in-vivo activity of CAR-T cells.

[0064] There were three different options for the transmembrane domain of the CAR22, i.e., CD8TM, CD28TM or ICOSTM, their amino acid sequences were as set forth in SEQ ID NO: 11, SEQ ID NO: 19 and SEQ ID NO: 25, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 26, respectively.

[0065] There were four different options for the co-stimulator domain of the CAR22, i.e, 4-1BBCSD, CD28CSD, ICOSCSD and OX40CSD, their amino acid sequences were as set forth in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 29, respectively; and the nucleotide sequences encoding the above-mentioned amino acid sequences were as set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 30, respectively. That is, the CAR22s in seven bispecific chimeric antigen receptor CAR19-CAR22s encoded the same antigen binding domain (i.e., having the same scFv) and CD3z signaling domain, wherein the amino acid sequence of the CD3z signaling domain was as set forth in SEQ ID NO: 15 (the nucleotide sequence encoding this amino acid sequence was as set forth in SEQ ID NO: 16). The only difference between the seven constructs was the different combinations of the hinge region, the transmembrane domain and the co-stimulator domain in CAR22s.

Example 2: Comparison of the Killing Effects of CAR19-CAR20-T Cells Prepared from Bispecific Chimeric Antigen Receptors with Different Structures on Target Cells

[0066] The CAR19-CAR20 combinational bispecific chimeric antigen receptors as described in Example 1 were used to prepare dual-targeting CAR-T cells, and then the dual-targeting CAR-T cells were co-incubated with two different kinds of target cells, i.e., CD19.sup.+K562-luc-GFP and CD20.sup.+K562-luc-GFP for 18 to 24 hours at different effector cell (E): target cell (T) ratios, that is, co-incubated at a E/T ratio of 1:1, 2.5:1, 5:1, 10:1 or 20:1, respectively. T cells without genetic modification (that is, T cells that had not been subjected to lentivirus infection, hereinafter referred to as NC-T cells) were used as the background control, the constructed target cell strain was labeled with luciferase, and the killing effects of effector cells on target cells were determined based on the principle of chemiluminescence. The specific operations were as follows.

[0067] (1) Isolation of PBMC from Peripheral Blood, Isolation and Activation of T Cells, Lentiviral Transduction and In-Vitro Culture

[0068] Healthy donors tested negative for HBV, HCV and HIV were selected, 100 ml of blood was drawn from the median cubital vein, PBMCs were isolated from buffy coat via Ficoll density gradient centrifugation, and the number of CD3.sup.+T cells were calculated according to the percentage of CD3.sup.+T cells determined via whole blood flow cytometry. The magnetic beads were aspirated in its using amount (DynaBeads CD3/CD28:CD3.sup.+T cell=3:1) and incubated with cells in the buffy coat for 30 min. CD3.sup.+T cells were isolated and activated by Dynabeads CD3/CD28 (Lifetechnologies, Cat. No.: 40203D) for 24 hours, followed by the determination of the proportion of CD25.sup.+CD69.sup.+ T cells via flow cytometry (the proportion of CD25.sup.+CD69.sup.+ T cell: 71%). CD3.sup.+ T cells were subjected to lentiviral transduction after activation. A Novonectin-coated 24-well plate was incubated at 37° C. for 2 hours, the cell suspensions obtained after the above operations were respectively formulated into transduction systems with each of the prepared lentiviruses (that is, lentiviruses respectively comprising PCTL126, PCTL137, PCTL138, PCTL139, PCTL151, PCTL152, and PCTL153) (MOI=8), Synperonic® F108 (Sigma, Cat. No.: 07579-250G-F, 10 μg/ml) and Tscm (2 U/ml), the transduction systems were charged in the coated 24-well plate, the cell density was adjusted to 1.0E+06 cells/ml, followed by centrifugation at 500 g for 30 min and subsequent static culture in an incubator containing CO.sub.2 at 37° C. for 48 h. After transfection, cells were cultured in X-vivo15 medium (LONZA, Cat. No.: 04-418Q) containing 5% FBS, Tscm (final concentration: 2 U/ml) was supplemented every other day, cell counting was conducted, the cell density was adjusted to 0.5E+06 cells/ml, and cells were harvested after being cultured to Day 8 to Day 10.

[0069] (2) Preparation of effector cells (dual-targeting CAR-T cells): (NC-T cells (T cells that had not been subjected to lentivirus infection) that had been proliferated for 5 to 7 days and CAR-T cells in each group were taken, followed by observation under a microscope to judge whether the growth status of cells was normal. @ NC-T cells and CAR-T cells in each group were collected into a 15-mL centrifuge tube or a 50-mL centrifuge tube, and the total number of cells was counted (Cellometer k2 cell counter). @ The collected cells were washed once or twice with sterile PBS (Hyclone, Cat. No.: SH30256.01) and centrifuged at 1500 rpm for 5 minutes at 25° C. @ The washed cell pellet was re-suspended with T cell culture medium X-VIVO15 (LONZA, Cat. No.: 04-418Q) (without autologous serum and IL-2), and the cell density was adjusted to 5.0E+07 cells/mL.

[0070] (3) Preparation of target cells: {circle around (1)} Target cells, i.e., CD19.sup.+K562-luc-GFP and CD20.sup.+K562-luc-GFP (Tsukahara et al. Biochem Biophys Res Commun. 2013; 438(1):84-89), were taken and observed under a microscope to judge whether the cell status was normal. {circle around (2)} The two kinds of target cells mentioned above were respectively collected into a 15-mL centrifuge tube or a 50-mL centrifuge tube, and the total number of cells was counted. {circle around (3)} The collected cells were washed once or twice with sterile PBS and centrifuged at for 5 minutes 1500 rpm at 25° C. {circle around (4)} The washed cell pellet was re-suspended with RPM11640 (gibco, Cat. No.: 11875-093) (without FBS), and the cell density was adjusted to 5.0E+06 cells/mL.

[0071] (4) In-vitro killing: {circle around (1)} Preparation of killing systems: In a 1.5-mL centrifuge tube, effector cells (i.e., NC-T cells and CAR-T cells in each group) with adjusted density were respectively mixed with target cells (i.e., CD19.sup.+K562-luc-GFP and CD20.sup.+K562-luc-GFP) at different effector-to-target ratios, specifically, effector cells (CAR-T cells) and target cells were respectively mixed at a ratio of 1:1, 2.5:1, 5:1, 10:1 or 20:1, and T cell culture medium X-VIVO15 (LONZA, Cat. No.: 04-418Q) (without autologous serum and IL-2) was added until the total volume was up to 200 μL; {circle around (2)} 200-μL killing systems prepared above were respectively transferred into a 96-well V-shape plate for co-incubation for 24 hours. {circle around (3)} After 24 hours, cells in each well of the 96-well V-shape plate were gently pipetted and mixed evenly, and 100 μL of cell suspensions were respectively transferred into a 96-well plate with white wall and non-transparent bottom. 100 μL of ONE-Glo™ Luciferase Assay Substrate was added, and chemiluminescence (Luminescence) was determined by Luminoskan Ascent chemiluminescence analyzer after the system was incubated in the dark at room temperature for 10 minutes.

Calculation of killing efficiency: Killing efficiency=(the corresponding value of NC-T cells−the value of specific CAR19-CAR20-T cell at corresponding effector-to-target ratio)/the corresponding value of NC-T cells

[0072] Experimental results: As could be seen from the results as shown in Table 3, in a case where the effector-to-target ratio was 10:1, CAR19-CAR20-T cells comprising one of the seven bispecific chimeric antigen receptors (that is, PCTL126, PCTL137, PCTL138, PCTL139, PCTL151, PCTL152 and PCTL153) had a killing efficiency of more than 90% for CD19+K562-luc-GFP target cells. Among them, CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 had a killing efficiency of approximately 100% for CD19.sup.+K562-luc-GFP target cells (please refer to Table 3 and FIG. 3 for details). In addition, CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 still had relatively high killing efficiency for the target cells in a case where the effector-to-target ratio was relatively low.

TABLE-US-00003 TABLE 3 Killing efficiency of bispecific chimeric antigen receptor CAR19-CAR20-T cells for CD19.sup.+K562-luc-GFP target cells 1:1 2.5:1 5:1 10:1 20:1 PCTL137 −273.22% 40.27% 94.13% 96.58% 98.60% PCTL138 −178.91% 75.38% 97.68% 98.31% 99.41% PCTL139 −153.56% 41.80% 96.77% 97.60% 98.72% PCTL126 −26.72% 87.59% 97.59% 98.95% 99.65% PCTL151 −28.95% 83.53% 93.95% 96.44% 99.07% PCTL152 40.19% 95.92% 99.12% 99.58% 99.85% PCTL153 43.95% 96.83% 97.71% 99.40% 99.72%

[0073] As could be seen from the results as shown in Table 4, in a case where the effector-to-target ratio was 20:1, CAR19-CAR20-T cells comprising one of the seven bispecific chimeric antigen receptors (that is, PCTL126, PCTL137, PCTL138, PCTL139, PCTL151, PCTL152 and PCTL153) had a killing efficiency of more than 80% for CD20.sup.+K562-luc-GFP target cells. Among these, CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 had a killing efficiency of approximately 100% for CD20.sup.+K562-luc-GFP target cells (please refer to Table 4 and FIG. 4 for details).

TABLE-US-00004 TABLE 4 Killing efficiency of bispecific chimeric antigen receptor CAR19-CAR20-T cells for CD20.sup.+K562-luc-GFP target cells 1:1 2.5:1 5:1 10:1 20:1 PCTL137 −269.38% −201.37% −2.41% 72.28% 81.53% PCTL138 −204.88% −49.82% 11.09% 71.10% 80.52% PCTL139 −279.06% −160.74% 13.09% 81.29% 86.59% PCTL126 −138.73% −33.19% 67.29% 71.61% 90.32% PCTL151 −136.73% −12.02% 45.50% 70.90% 88.66% PCTL152 −182.88% 15.71% 75.81% 92.37% 98.25% PCTL153 −161.64% −32.69% 81.21% 89.58% 96.97%

Example 3: T Cell Phenotypes Exhibited by CAR19-CAR20-T Cells Prepared from Bispecific Chimeric Antigen Receptors with Different Structures

[0074] Dual-targeting CAR-T cells were prepared using the CAR19-CAR20 combinational bispecific chimeric antigen receptors as described in Example 1, and differentiated cell populations were analyzed by a flow cytometer using conventional T cell differentiation antigens and antibodies on Day 7 to Day 10 after lentiviral transfection.

[0075] Experimental methods: 7 kinds of bispecific chimeric antigen receptor CAR19-CAR20s prepared in Example 1 were selected as experimental materials, and the corresponding dual-targeting CAR-T cells were prepared according to the preparation method of effector cells as described in Example 2. For each of the 7 kinds of dual-targeting CAR-T cells as prepared, 1×10.sup.6 CAR-T cells were taken, the CAR-T cells were washed with PBS and then incubated with CD62L-PE-Cy5 antibody (BD, Cat. No.: 555545) and CD45RO-FITC antibody (BD, Cat. No.: 555492) in a freezer at 4° C. for 30 min. After the completion of the incubation with antibodies, the resultant was washed with PBS (Hyclone, Cat. No.: SH30256.01) 2-3 times, re-suspended with 500 μl of PBS, and then placed in a flow cytometry tube to prepare for determination on a flow cytometer.

[0076] Experimental results: As shown in FIG. 5, the results indicated that all the analyzed dual-targeting CAR-T cells were capable of maintaining a relatively high proportion of stem cell-like central memory T cells (TSCMs). Studies had reported that the proportion of the stem cell-like central memory T cell population was closely related to the tumoricidal activity, proliferating ability and lasting immunological memory of CAR-T cells in organisms. This suggested that the dual-targeting CAR-T cells provided in the present disclosure were capable of having relatively good tumoricidal activity, proliferating ability and lasting immunological memory in organisms.

Example 4: Detection of the Expression of CAR19 and CAR20 in CAR19-CAR20-T Cells Comprising PCTL152 and CAR19-CAR20-T Cells Comprising PCTL153

[0077] Experimental methods: CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 with relatively high killing efficiency in Example 2 were selected as experimental materials. For each of the two kinds of CAR-T cells mentioned above, 1×10.sup.6 CAR-T cells were taken, washed with 4% BSA (2500 rpm, 5 min) three times, and then incubated with antibodies as follows. (1) Cells were incubated with Alexa Fluor 647 AffiniPure Goat Anti-Human IgG (1:100 to 1:800) in a freezer at 4° C. for 30 min. After the completion of the incubation with the antibody, the resultant was washed with 4% BSA (2500 rpm, 5 min) three times, and then incubated with the following antibody, i.e., (2) PE-labeled CAR19 (iFMC63) idiotype (Qin et al. Mol Ther Oncolytics. 2018; 11: 127-137) (1 μg/ml) in a freezer at 4° C. for 30 min. After the completion of the incubation with the antibody, the resultant was washed with 4% BSA 2-3 times (2500 rpm, 5 min), re-suspended with 500 μl of PBS, and then placed in a flow cytometry tube to prepare for determination on a flow cytometer.

[0078] Experimental results: As shown in FIG. 6, both CAR19 protein and CAR20 protein could be simultaneously detected on the surface of T cells. The results indicated that the constructed CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 were capable of expressing CAR19.sup.+CAR20.sup.+ dual-positive population well.

Example 5: Validation of the In-Vivo Tumor Inhibitory Activity of CAR19-CAR20-T Cells Comprising PCTL152 and CAR19-CAR20-T Cells Comprising PCTL153 in Mice

[0079] Experimental methods: CAR19-CAR20-T cells comprising PCTL152 and CAR19-CAR20-T cells comprising PCTL153 with relatively high killing efficiency in Example 2 were selected as the experimental groups, and PBS was selected as the control group. Raji-Luc cells (Biocytogen, Cat. No.: B-HCL-010) re-suspended with PBS were inoculated into B-NDG® (B-NSG) mice with a concentration of 5×10.sup.5 cells/0.2 mL and a volume of 0.2 mL/mice by intravenous injection via tail vein. On the day of inoculation, a small animal imaging system was utilized to observe whether the tumor inoculation was successful. On Day 3 after inoculation, tumor growth was measured by using the small animal imaging system. When the average imaging signal reached approximately 1×10.sup.6 [(P/S)/(cm.sup.2/sr)], 8 mice with moderate tumor imaging signal were selected and enrolled, and were randomly assigned to 3 groups (2 mice in Group G1, 3 mice in each of Group G3 and Group G4). Mice showing excessively strong/excessively weak fluorescence signal were excluded. Administration began on the day of grouping, and tumor growth (detected and recorded by the small animal imaging system) was detected and the body weights of the animals were measured on Day 4 after administration. Afterwards, mice were detected on the imaging system once a week (Day 4, Day 11, Day 18 and Day 25) and the body weights of the animals were measured twice a week. The specific dosage regimen was as shown in FIG. 7. Please refer to Table 5 for the specific details of the type of the administered substances, the number of mice and the dosage of administration in each group.

TABLE-US-00005 TABLE 5 Type of the administered substances, number of mice and dosage of administration of Group G1, Group G3 and Group G4 Type of the administered Number Group substances of mice Dosage of administration G1 PBS 2 PBS 200 μl/mice G3 PCTL152 3 Total T 0.1E+07/200 μl/mice G4 PCTL153 3 Total T 0.1E+07/200 μl/mice

[0080] Experimental results: Up to Day 28, all the mice in Group G1 and Group G3 died, and the survival rate of the mice in Group G4 was 67.7%. It could be seen that, as compared with CAR19-CAR20-T cells comprising PCTL152, CAR19-CAR20-T cells comprising PCTL153 were capable of increasing the survival rate of the tumor-bearing mice significantly (please refer to FIG. 8).

Example 6: Validation of the In-Vivo Tumor Inhibitory Activity of CAR19-CAR20-T Cells Comprising PCTL153, Single-Targeting CAR19-T Cells, Single-Targeting CAR20-T Cells and Tandem CAR20-19-T Cells in Mice

[0081] Experimental methods: Raji-Luc cells re-suspended with PBS were inoculated into B-NDG® (B-NSG) mice with a concentration of 5×10.sup.5 cells/0.2 mL and a volume of 0.2 mL/mice by intravenous injection via tail vein, and 54 mice were inoculated in total. On the day of inoculation, a small animal imaging system was utilized to observe whether the tumor inoculation was successful. Tumor growth was measured by using the small animal imaging system after the successful inoculation. When the average imaging signal reached approximately 1×10.sup.6[(P/S)/(cm.sup.2/sr)], 30 mice with moderate tumor imaging signal were selected and enrolled, and were randomly assigned to 5 groups (6 mice per group). Tumor-bearing mice showing excessively strong/excessively weak living imaging signal were excluded. Administration began on the day of grouping, the body weights of the experimental animals and the tumor growth (detected and recorded by the small animal imaging system) were continuously observed after administration. Tumor growth was measured on Day 4, Day 7 and Day 11 after grouping, afterwards, tumor growth was measured once a week (detected and recorded by the small animal imaging system). The body weights of the animals were measured twice a week, clinical observation was performed, and the measured values were recorded. The specific dosage regimen was as shown in FIG. 9. Please refer to Table 6 for the specific details of the type of the administered substances, the number of mice and the dosage of administration.

[0082] Experimental results: As compared with the traditional single-targeting CAR19-T cells (Group G3, the structure and sequence of the CAR19 comprised therein were the same as those of the CAR19 described in Example 1 of the present disclosure, i.e., FMC63-CD8 hinge-CD8 TM-4-1BB-CD3z), single-targeting CAR20-T cells (Group G4, the structure and sequence of the CAR20 comprised therein were the same as those of the CAR20 in the PCTL153 described in Example 1 of the present disclosure, i.e., Leu16-IgG4mt10+N297A hinge-ICOSTM-ICOSCSD-CD3z) and tandem CAR20-19-T cells (Group G7, CD20 scFv-(EAAAK)3-CD19 scFv-IgG4 hinge-CD28 TM-4-1BB-CD3z, please refer to Zah et al. Cancer Immunol Res. 2016; 4(6):498-508 for details), the combinational dual-targeting CAR19-CAR20-T cells comprising PCTL153 (Group G5) were capable of increasing the survival rate of tumor-bearing mice significantly (please refer to FIG. 10).

TABLE-US-00006 TABLE 6 Type of the administered substances, number of mice and dosage of administration of Group G1, Group G3, Group G4, Group G5 and Group G7 Type of the administered Number Group substances of mice Dosage of administration G1 PBS 6 PBS 200 μl/mice G3 CAR-19-T 6 Total T 1.0E+07/200 μl/mice G4 CAR-20-T 6 Total T 1.0E+07/200 μl/mice G5 PCTL153 6 Total T 1.0E+07/200 μl/mice (combinational CAR19-CAR20) G7 tandem CAR20- 6 Total T 1.0E+07/200 μl/mice CAR19

Example 7: Comparison of the Killing Effects of CAR19-CAR22-T Cells Prepared from Bispecific Chimeric Antigen Receptors with Different Structures on Target Cells

[0083] The combinational bispecific chimeric antigen receptor CAR19-CAR22s as described in Example 1 were used to prepare dual-targeting CAR-T cells, and then the dual-targeting CAR-T cells were co-incubated with two different kinds of target cells, i.e., CD19.sup.+K562-luc-GFP and CD22.sup.+K562-luc-GFP for 18 to 24 hours at different effector cell (E): target cell (T) ratios, that is, at a E/T ratio of 5:1, 10:1 or 20:1, respectively. T cells without genetic modification (that is, T cells that had not been subjected to lentivirus infection, hereinafter referred to as NC-T cells) were used as the background control, the constructed target cell strain was labeled with luciferase, and the killing effects of effector cells on target cells were determined based on the principle of chemiluminescence. The specific operations were as follows.

[0084] (1) Isolation of PBMC from peripheral blood, isolation and activation of T cells, lentiviral transduction and in-vitro culture

[0085] Healthy donors tested negative for HBV, HCV and HIV were selected, 100 ml of blood was drawn from the median cubital vein, PBMCs were isolated from buffy coat via Ficoll density gradient centrifugation, and the number of CD3.sup.+T cells were calculated according to the percentage of CD3.sup.+ T cells determined via whole blood flow cytometry. The magnetic beads were aspirated in its using amount (DynaBeads CD3/CD28:CD3.sup.+ T cell=3:1) and incubated with cells in the buffy coat for 30 min. CD3*T cells were isolated and activated by Dynabeads CD3/CD28 (Lifetechnologies, Cat. No.: 40203D) for 24 hours, followed by the determination of the proportion of CD25.sup.+CD69.sup.+ T cells via flow cytometry (the proportion of CD25.sup.+CD69.sup.+ T cell: 71%). CD3.sup.+ T cells were subjected to lentiviral transduction after activation. A Novonectin-coated 24-well plate was incubated at 37° C. for 2 hours, the cell suspensions obtained after the above operations were respectively formulated into transduction systems with each of the prepared lentiviruses (that is, lentiviruses respectively comprising PCTL81, PCTL103, PCTL105, PCTL124, PCTL148, PCTL149, and PCTL150) (MOI=8), Synperonic® F108 (Sigma, Cat. No.: 07579-250G-F, 10 μg/ml) and Tscm (2 U/ml), the transduction systems were charged in the coated 24-well plate, the cell density was adjusted to 1.0E+06 cells/ml, followed by centrifugation at 500 g for 30 min and subsequent static culture in an incubator containing CO.sub.2 at 37° C. for 48 h. After transfection, cells were cultured in X-vivo15 medium (LONZA, Cat. No.: 04-418Q) containing 5% FBS, Tscm (final concentration: 2 U/ml) was supplemented every other day, cell counting was conducted, the cell density was adjusted to 0.5E+06 cells/ml, and cells were harvested after being cultured to Day 8 to Day 10.

[0086] (2) Preparation of effector cells (dual-targeting CAR-T cells): {circle around (1)} NC-T cells (T cells that had not been subjected to lentivirus infection) that had been proliferated for 5 to 7 days and CAR-T cells in each group were taken, followed by observation under a microscope to judge whether the growth status of cells was normal. {circle around (2)} NC-T cells and CAR-T cells in each group were collected into a 15-mL centrifuge tube or a 50-mL centrifuge tube, and the total number of cells was counted (Cellometer k2 cell counter). {circle around (3)} The collected cells were washed once or twice with sterile PBS (Hyclone, Cat. No.: SH30256.01) and centrifuged at 1500 rpm for 5 minutes at 25° C. {circle around (4)} The washed cell pellet was re-suspended with T cell culture medium X-VIVO15 (LONZA, Cat. No.: 04-418Q) (without autologous serum and IL-2), and the cell density was adjusted to 5.0E+07 cells/mL.

[0087] (3) Preparation of target cells: {circle around (1)} Target cells, i.e., CD19.sup.+K562-luc-GFP and CD22.sup.+K562-luc-GFP (Tsukahara et al. Biochem Biophys Res Commun. 2013; 438(1):84-89), were taken and observed under a microscope to judge whether the cell status was normal. {circle around (2)} The two kinds of target cells mentioned above were respectively collected into a 15-mL centrifuge tube or a 50-mL centrifuge tube, and the total number of cells was counted. {circle around (3)} The collected cells were washed once or twice with sterile PBS and centrifuged at 1500 rpm for 5 minutes at 25° C. {circle around (4)} The washed cell pellet was re-suspended with RPMI1640 (gibco, Cat. No.: 11875-093) (without FBS), and the cell density was adjusted to 5.0E+06 cells/mL.

[0088] (4) In-vitro killing: {circle around (1)} Preparation of killing systems: In a 1.5-mL centrifuge tube, effector cells (i.e., NC-T cells and CAR-T cells in each group) with adjusted density were respectively mixed with target cells (i.e., CD19.sup.+K562-luc-GFP and CD22.sup.+K562-luc-GFP) at different effector-to-target ratios, specifically, effector cells (CAR-T cells) and target cells were respectively mixed at a ratio of 5:1, 10:1 or 20:1, and T cell culture medium X-VIV015 (LONZA, Cat. No.: 04-418Q) (without autologous serum and IL-2) was added until the total volume was up to 200 μL. {circle around (2)} 200-μL killing systems prepared above were respectively transferred into a 96-well V-shape plate for co-incubation for 24 hours. {circle around (3)} After 24 hours, cells in each well of the 96-well V-shape plate were gently pipetted and mixed evenly, and 100 μL of cell suspensions were respectively transferred into a 96-well plate with white wall and non-transparent bottom. 100 μL of ONE-Glo™ Luciferase Assay Substrate was added, and chemiluminescence (Luminescence) was determined by Luminoskan Ascent chemiluminescence analyzer after the system was incubated in the dark at room temperature for 10 minutes.

Calculation of killing efficiency: Killing efficiency=(the corresponding value of NC-T cells−the value of specific CAR19-CAR22-T cell at corresponding effector-to-target ratio)/the corresponding value of NC-T cell

[0089] Experimental results: As could be seen from the results as shown in FIG. 11, in a case where the effector-to-target ratio was 10:1, CAR19-CAR22-T cells comprising one of the seven bispecific chimeric antigen receptors (that is, PCTL81, PCTL103, PCTL105, PCTL124, PCTL148, PCTL149 and PCTL150) had a killing efficiency of more than 90% for CD19.sup.+K562-luc-GFP target cells (please refer to FIG. 11 for details). As could be seen from the results as shown in FIG. 12, all the CAR19-CAR22-T cells comprising one of the seven bispecific chimeric antigen receptors (that is, PCTL81, PCTL103, PCTL105, PCTL124, PCTL148, PCTL149 and PCTL150) had killing effects on CD22.sup.+K562-luc-GFP target cells and were apparently effector-to-target ratio-dependent (please refer to FIG. 12 for details).